O101-S Examination for FOB in the common marmoset

1 2 2 2 ○Inoue Ryo ,Atsushi FUJIWARA ,Kazue ARUGA ,Masahiko IINO ,Chiyoko NISHIME1,Eiko NISHINAKA1,Shinichi SATO2 1Central Institute for Experimental Animals 2Ina Research Inc

Purpose:The common marmoset (marmoset) is small primates, which their body weights are about the same as rats. Functional Observational Battery (FOB) is a test to evaluate the effect of a compound on the central nervous systems (CNS) in drug development. In this study, we investigated if the marmoset is useful to possess high-sensitivity primate-specific FOB.Methods:Saline, Cocaine (Coc 2 or 1 mg/kg) or Chlorpromazine (Cpz 3 or 1 mg/kg) was intravenously administered to four male marmosets. FOB (general condition, motor function, etc.) was performed 5 minutes to 6 hours after the administration.Results:Rapid and Slight movements of the head which is sepcific for the non-human primate, continual movement, hyperirritability and hyperthermia were observed at 5 min after administration in Coc 2 mg/kg group. Although continual movement, hyperirritability and hyperthermia were also observed in Coc 1 mg/kg group, expression of other symptoms were decreased. Eye-closing, hypoactivity, slowed motion and ataxia were observed at 5 min after administration of Cpz 3 mg/kg. Eye-closing, hypoactivity were also observed in Cpz 1 mg/kg group, but expression of other symptoms were decreased.Discussion:Dose dependent effects of Coc and Cpz on the CNS were able to be detected in the marmoset FOB. Since the primate-specific symptom such as rapid and slight of the head movement was observed in the Coc treatment, FOB in the marmosets may be more useful than that in rats.

1P001-T Introduction of Institute of Laboratory Animal Research, Nagoya University

○Mariko Itoh,Rarami Mori,Mayumi Toda,SHuichi Aono,Jun Sato

Center for Animal Research and Education, Nagoya University, Nagoya, Japan

In Higashiyama area of Nagoya University, Institute of Laboratory Animal Facility, a shared new SPF (specific pathogen free) facility, has opened. It is a three-story facility with total floor area of 2,218 m2 for rats and mice. By this facility, we efficiently centralized the management of animals in Higashiyama area, and established a safer and more appropriate environment for animal experimentation.

S 87 1P002-T The large-scale repair and renovation work conducted in animal facility of Niigata University (3) 1 1 1 1 ○Yoshitaka MAEDA , Kanako ODA , Seiko SAKAI , Fumiya NAGATA , Yukihiro JIMBO1, Minoru TANAKA1, Yoshitaka YAMAMOTO1, Saori CHIBA1, Toshiya SATO1, Nobuyoshi FUJISAWA1, Minesuke YOKOYAMA1,2, Toshikuni SASAOKA1 1) Animal Resources Branch Department of Comparative & Experimental Medicine Brain Research Institute, Niigata University, Niigata, Japan 2) Central Institute for Experimental Animals, Kawasaki, Japan

The animal facility of Brain Research Institute, Niigata University conducted a major building renovation over a period of seven months from January 2013. Last year in the Annual Meeting of the Japanese Association for Experimental Animal Technologists, we reported two papers entitled "Shutdown and transfer of the animal facility" and "Management of temporary facility and re-launch of the new facility." In the present paper, we will report the experience in the process of resuming our facility.

1P003-T Aseismic measures in the new laboratory animal facility

○Satomi Takano, Kaori Muguruma, Noboru Ogiso

Laboratory of Research Animal, National Center for Geriatrics and Gerontology, Aichi, Japan

Earthquake measures have been implemented in research institutions and universities from the lessons of the Great East Japan Earthquake and the Great Hanshin Earthquake. In the new laboratory animal facility which was completed in September 2012, we try to carry out seismic measures in NCGG with experts. Most of aseismic fixing method was need to drill a hole in the floor or walls so far. But this method is not necessarily suitable to the laboratory and breeding room because cleaning and disinfection of the day-to-day is difficult. Recently, the seismic fixing bracket which combines earthquake-resistant mat and metal parts was developed. This fixing bracket does not need holes in the floor or walls. In this study, we examined whether or not this fixing bracket is useful in laboratory and breeding room. We were attached the seismic fixing bracket to individual ventilated breeding rack and gas cylinder mount. We were compared with before mounting for deterioration of the mat cleanliness and workability and, of seismic safety equipment for the installation location. The cylinder frame, there was no effect on the vibration rather than hinder the work of the cylinder desorption. We are currently studying the cleanliness measurement of seismic equipment and work due to desorption of the fixing bracket.

S 88 1P004-T Efforts of Laboratory Animals Managers in AAALAC International accreditation

○Kazutaka Fujita, Kosaka Mitsuhiro, Yoshio Nemoto, Yukari Nishio, Kinya Fujii, Tsuyoshi Mizoguchi, Muneo Haraguchi, Shizuka Nitta, Masanori Kanda Shionogi TechnoAdvance Research Co., Ltd

In receiving AAALAC International accreditation, our laboratory animal manager has been largely involved in three major points as follows: identification of issues and revision of operating procedures, introduction of veterinary care, and environmental enrichment. Through these activities, we reaffirmed important roles of the laboratory animal manager, and animal observation and facility management were centered on the animal. Future subjects will be also discussed.

1P005-T Construction of rearing management system with bar-code

○Chika Uefuji, Norio Yata, Kazutaka Ueyama, Masayuki Arakawa, Masahiro Fujii, Haruko Hirayama, Katsumi Mominoki Department of Animal Resources, Advanced Science Research Center, Okayama University, Okayama, Japan

To efficiently manage the animal facility, it is important that the quantity of the laboratory animals, especially transgenic mice, have been a handle on. However, there are too many difficulties, time-consuming, in doing this work without a support system for the management of the animals. Although there have been commercially available ones, these initial installations are more expensive. Moreover, these systems are less flexible under saving the cost. Therefore, we have been trying to come up with the low-cost and flexible system using a general-purpose component such as King Jim Tepra Pro, and a widely used software with the visual basic for application (VBA), such as Microsoft Excel. If the computer network has already been established in the facility, the initial cost is less than 150,000 Yen per one package, includes a tablet type personal computer with the required software, a bar cord reader and a label printer. Namely, VBA allows our developed system to easily, cheaply and unlimitedly expand the specifications in proportion to the increase in the volume of information. The high flexibility of our system has been helpful to easily get a handle on the quantity of the animals in our facility, so that we propel the concept of three Rs, especially "Reduction" , in our facility.

S 89 1P006-S Development of Web-Based Laboratory Animal Care System 1 1 1 1 1 ○Jun Sato ,Shuichi Aono ,Akiko Tomotsugu ,Mariko Itoh ,Rarami Mori ,Mayumi Toda1,Nobuya Shosaki2 1Center for Animal Research and Education, Nagoya University, Nagoya, Japan 2EVANEXT limited partnership company, Kakogawa, Hyogo, Japan

There are many rearing management items in animal experiments, and in order to support the smooth operation of the laboratory animal facility, it is necessary to develop an optimal management system. However, since the existing systems are expensive, it does not promote the introduction of a software system at universities or research institutes. Although the introduction of the paper-based system is a cost-cutter, it takes up a lot of time and work to convert paper-based information to digital format. Moreover, it is not desirable for hygienic environment. We developed a web-based laboratory animal care system and have been innovating the developed system since July 2013. Users can feed information to diary management and husbandry from a tablet computer at the animal rooms, and send animal's information in real time to a Web server. The management states and the animal experimental plan are accessible from a computer with permission connected to the Internet. By storing in the database automatically, we reduce the time and effort spent to tallying process. Furthermore, fees and billing are automatically calculated based on the information obtained from the diary management and husbandry. As a future subject, we would like to provide guidance about information moral and strengthen information security.

1P007-T Significance of microbial monitoring in the animal facility environment: Part 2 1 2 2 ○Yuko Moriya ,Syuki Ojima , Ryuichiro Ando

1JAC, Inc., Tokyo, Japan 2 Center for Laboratory Animal Science, Tohoku Pharmaceutical University, Sendai, Japan

In order to grasp if the facility environment is properly maintained, we performed setting plate sampling before and after cleaning and disinfecting the floor. Results of the comparative analysis on an annual basis, the number of bacteria was reduced after disinfection and a significant difference was observed in 10 of 23 areas. In order to maintain proper environment, the time and frequency of the cleaning and disinfection should be considered.

S 90 1P008-S Evaluation experiment of indoor decontamination using hydrogen peroxide vapor

○Satoshi Saitou,Kentaro Amano

Takenaka Corporation, Research & Development Institute, Inzai, Chiba, Japan

[Purpose] As a method of decontamination of a laboratory-animal breeding room, formaldehyde fumigation has been used, but the regulation of formaldehyde becomes severe, so the drug has gone out of use year by year. We report the experiments of decontamination using hydrogen peroxide.[Methods] We carried out the experiments in a biological clean room (BCR). The concentration in the air was measured using a gas detection system, and germicidal effect was evaluated by biological indicator (BI) or chemical indicator. As for the condition of decontaminating, 3 cases (case I; low concentration - high humidity, case II; low concentration - low humidity, case III; high concentration - low humidity) were set up. [Results and discussions] All BIs in the 3 cases at corner parts of the BCR, back of the safety cabinet etc, showed culture negative. But some BIs inside the air suction ports of safety cabinet, and under the punching panel showed culture positive. Because these points were spaces of the form of small gap, it is thought that there is little replacement of the air, and hydrogen peroxide vapor was hard to arrive. From the results of germicidal and corrosive effects, we concluded that the case II was superior.[Acknowledgement] We are grateful to Hiroyuki Kato, Toshihiro Asano, Kosuke Kuwahara, Akinobu Oishi belonging to Daiwa Can Campany General Laboratories, and Hisao Watanabe belonging to YMP International Corporation, for providing a hydrogen peroxide vapor generator.

1P009-T Evaluation of aseptic conditions for sterile materials stored in sterilization bag

○Satomi Mizutani, Kouhei Higashiyama,Shinobu Ohba,Yuji Sudo,Hironari Koyama

Astellas Research Technologies Co., Ltd

Laboratory Animal Science Div.,Astellas Research Technologies Co.,Ltd., Objective: To evaluate the condition of sterile samples used in our animal breeding facilities after sterilization, a sterility test was performed using sterile materials at different store periods. Methods: Four sample materials made of metal, silicone rubber, plastic, and paper were inserted into individual sterilization bag. Samples sterilized in an autoclave were kept in a controlled temperature (20-26 C) and humidity (40-70 %) environment for 13, 14, 15, and 24 months. A sterility test was performed in accordance with the Japanese Pharmacopoeia, 16th edition. Two sterilized culture media, Soybean-Casein Digest medium (SCD) and Thioglycollate medium (TGC), were used for sterility cultures. Results: Sterility cultures in 10 of 12 samples were negative for bacterial growth at 13 months after sterilization. Sterility cultures in 2 other samples yielded Staphylococcus epidermidis at the same time point. A new sterility test improved over the initial regimen by adding a sterilization-by-fire step was conducted on samples of every material. All sterility cultures of the 96 samples were negative for bacterial growth at 14-24 months after sterilization. Conclusion: Our findings suggest that sterile materials remain aseptic for at least 24 months, namely 2 years, after sterilization.

S 91 1P010-S Mycoplasma hyorhinis degrades amyloid-β peptides

1 2 1 ○Koyama Naoki ,Jun Tan ,Takashi Mori

1Department of Biomedical Sciences, Saitama Medical Center/University 2Department of Psychiatry & Behavioral Neurosciences, University of South Florida

Alzheimer's disease (AD) is the most common dementia and is a growing worldwide public health concern. Accumulation of amyloid-β (Aβ) peptides and their aggregation into plaques in the brain are thought to be the one of the major causes of AD. Despite continued efforts from academia and the pharmaceutical industry, no new drugs for AD have been developed since 2003. Using the Chinese hamster ovary cell line stably expressing human wild-type amyloid precursor protein, we found that Mycoplasma selectively degrades soluble Aβ in a time and dose (colony forming unit) dependent manner. Moreover, we fully characterized the Mycoplasma species as Mycoplasma hyorhinis (M. hyorhinis) by genetic and colony morphological analyses. Most interestingly, we attenuated the pathogenicity of M. hyorhinis by γ irradiation (3.5 Gy), and found that its ability to degrade Aβ was retained. On the other hand, heated and/or sonicated M. hyorhinis failed to retain this ability to degrade Aβ, suggesting that this degradation requires viable microorganisms and likely a biologically active signaling pathway. In addition, we found that M. hyorhinis can degrade Aβ produced in AD model mice (PSAPP mice) ex vivo. Finally, we found that irradiated (non-pathogenic) M. hyorhinis also can degrade Aβ produced in PSAPP mice in vivo. These studies suggest that irradiated (non-pathogenic) M. hyorhinis can be a novel and alternative biological strategy for AD treatment.

1P011-S Cold-transport system for cauda epididymides taken from genetically engineered mouse at CARD

1 1,2 1,2 1,2 ○Fumi Takahashi ,Kiyoko Fukumoto ,Yukie Haruguchi ,Tomoko Kondo ,Yumi Takeshita1,2,Yuko Nakamuta1,2,Tomoko Umeno1,2,Mari Iwamoto1,Eri Kohagura1,Shuuji Tsuchiyama1,Toru Takeo1,Naomi Nakagata1 1Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto, Japan 2Kyudo Co., Ltd., Tosu, Japan

Our center has provided a cold-transport service for cauda epididymis taken from genetically engineered mice (GEM) since 2010. The cold transport of epididymides is a desirable means of shipment from the viewpoints of preventing microbial infections, eliminating the risk of mouse escape of death during transport, suppressing shipment cost and complying with animal welfare regulations. At the receiving facility, sperm collected from the transported epididymides can be used to carry out sperm cryopreservation and in vitro fertilization (IVF) for producing embryos to archive or expand a GEM colony. The average fertilization rate in IVF using sperm from cold-transported epididymides was 70-80%. In summary, the cold-transport system of cauda epididymides is a highly beneficial method of shipping GEM between research institutes.

S 92 1P012-S Introduction of mouse bank service in CARD, Kumamoto University via online videos 1 1,2 1,2 1,2 ○Mari Iwamoto ,Kiyoko Fukumoto ,Yukie Haruguchi ,Tomoko Kondo ,Yumi Takeshita1,2,Yuko Nakamuta1,2,Tomoko Umeno1,2,Fumi Takahashi1,Eri Kohagura1,Shuuji Tsuchiyama1,Toru Takeo1,Naomi Nakagata1 1 Division of Reproductive Engineering, Center for Animal Resources and Devel opment (CARD), Kumamoto University,Kumamoto, Japan 2Kyudo Co., Ltd., Tosu, Japan

Our center has provided a mouse bank service to support scientific research using genetically engineered mice (GEM) for researchers. In the mouse bank service, we apply various mouse reproductive techniques to effectively archive, produce and distribute GEM. We provide researchers with two different kinds of mouse bank service: a standard mouse bank plan and a premium mouse bank plan. In the standard plan, researchers can deposit their mice for free to allow for sharing among the scientific community. All information related to the deposited mice is published on the CARD R-BASE website, and anyone can easily obtain those mice by signing an MTA. Meanwhile, the premium plan allows researchers to privately store their mice. We also offer technical assistance to premium plan researchers for the efficient management of the preservation, reanimation and transport of stored mice. Recently, we have produced online videos (Japanese and English versions) which introduce our mouse bank. In this presentation, we will introduce the videos and the practical use of our service.

1P013-T An examination of woody enrichment in mice

○Naoki Ihara, Masanobu Yoshida, Tetsuya Sato, Katsuji Nakamura, Hirohiko Goto

Otsuka Pharmaceutical Co., Ltd., Department of Toxicology

[PURPOSE] As a part of the examination of environmental enrichment in mice, effect of providing a woody enrichment was examined. [METHODS] For individually housed ICR mice in steel cage, following enrichment was respectively provided for 1 to 4 weeks. A group; a woody stick (100×20×20mm), B group; 4 woody sticks, C group; a woody tunnel (100×40×40mm with a hole of 30mm in diameter), D group; a woody block consisted by 4 sticks. Another group was given a woody stick during the fasting time. [RESULTS AND DISCUSSION] In A and B groups, most of the mice showed decrease in the weight of their woody stick. In C and D groups, most of the tunnels and blocks were gnawed but some of them, which occurred in males dominantly, accompanied by the weight increment with urine and unusual smell. In male mice, a phenomenon called urinary scent marking has been reported. A tunnel or a block might be recognized as a part of their territory. Therefore, large-sized woody enrichment for mice was suggested to be needed periodical exchange. Under fasting condition, a stick weight reduced remarkably and fibrous materials were observed in 16 stomachs of 24 mice. For preventing ingestion of woody-material under fasting condition, removal of enrichment is recommended.

S 93 1P014-T Study of bedding material suitable for long-term breeding for aging laboratory animals 1 2 2 2 ○Tetsuya Omae , Noboru Ogiso , Satomi Takano , Kaori Muguruma

1KAC Corporation, Kyoto, Japan Laboratory of Research Animal, National Center for Geriatrics and Gerontology, 2Aichi, Japan At the National Center for Geriatrics and Gerontology (NCGG), genetically modified animals as well as non-modified aging animals are maintained. Because it is known that animal longevity is strongly influenced by the environment and management of the facility, we revised the breeding environment of aging animals to resume animal facility, and examined the bedding material.

1P015-T Trial of transition to Group Housing for Dogs 1.Evaluation of Connectable Cages

1,2， 1,2， 1,2， 1,2， ○Shoichi Sakurai Yasunori Nakayama Tatsuya Kusunoki Yuko kojima Atsushi Takai1,2, Kyoka Sudo1,2，Erika Unno1,2，Hiroko Nakano1 1Pharmaceutical Research and Technology Labs. Astellas Pharma Inc. Shizuoka, Japan 2JAC Co., Ltd. Shizuoka, Japan

In order to perform group housing of dogs without reducing the capacity within a limited space, we developed a new cage system that is equipped with sliding doors and ligatable to the currently owned two story cages. Because dogs were injured by projections inside of the cages, we have repaired cages and allowed dogs group-housed safely.

S 94 1P016-T The difference of the temperature and the humidity between inside the cage and in the animal room

○Ai Dantsuka, Natsumi Izawa, Tomoyuki Miura

Institute for Virus Research, Kyoto University, Kyoto, Japan

In our facility, we can conduct BSL-3 experiments using NHP. In this year, we installed a large-scale isolator that can keep up to eight monkeys. Its case size meets the Group 3 size of the recommendation of ILAR Guide and can also used for group 4 size cages. As these cages are individually ventilated and isolated, there is a possibility that the environment inside the cages is different from that in the animal room. Therefore, we measured the temperature and the humidity inside the cages and compared them to these in the animal room. The temperature in the cage was 1-2°C higher than that in the animal room. There wasn't significant difference about the humidity. These results suggest that it is necessary to maintain the room temperature in the animal room lower than usual to make the environment where monkeys are appropriate.

1P017-S The special food for rhesus macaque losing their appetite

○Ai Dantsuka,Natsumi Izawa,Tomoyuki Miura

Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, Kyoto, Japan

In our facility, monkeys often lose their appetite and body weight due to experimental procedures or environmental changes. Taking enough amount of food can lead to gain their body weight and reduce their physical demands. Therefore, we made the special food using their dairy diet and assessed whether it has effect on increasing their appetite. First, we grounded Primate Diet #5048 to a powder. Then, we mixed that powder with bananas and some fruit juice. After that, we froze them in an ice cube tray. By freezing, we can store the special food for about two weeks. We fed this special food to two monkeys. We fed the special food five days per week. We gradually decreased the number of days we fed the special food as the monkeys gain their appetite. And, we calculated the percentage of the amount of the food they took. Both monkeys ate the special food well, and they maintained better appetite even after they got some experimental procedure. The percentage of the food intake was higher when we fed the special food two or three days per week than five days per week. The special food successfully increased monkey's appetite and body weight. We think the monkeys prefer the special food because that was sweeter than the usual one because that contains bananas and fruit juice. We also think the reason for they increased their appetite for the usual Primate Diet #5048 is that the variation of food contributed as some kind of environmental enrichment.

S 95 1P018-S Combining isoflurane anaeshtesia with midazolam and butorphanol in mice

○Atsushi Tsukamoto,Mami Iimuro,Tomo Inomata Laboratory of Laboratory Animal Science, Azabu University, Kanagawa, Japan

Isoflurane is representative inhalant anaesthesia in laboratory animals. However, this drug mediates respiratory depression and clinical adverse reaction during induction. In the present study, we established novel balanced anaesthesia, combining isoflurane anaesthesia with midazolam and butorphanol (MB) in mice. Thirty-two male C57/BL6J mice were used. Animals received either isoflurane monoanaesthesia or isoflurane with MB (midazolam: 3 mg/kg, ip, butorphanol: 4 mg/kg, ip) premedication. Firstly, we evaluated minimum alveolar concentration (MAC) in each group by using bracketing method. Induction time and clinical adverse reaction were recorded in each group. Core body temperature, pulse rate, respiratory rate, and saturation O2 (SPO2) were assessed before and after induction for 1hr. Premedication with MB achieved 44% reduction of MAC, compared with that of isoflurane monoanaesthesia (Isoflurane monoanaeasthesia: 1.38±0.15 %, Isoflurane with MB: 0.78±0.10 %). When MB was premedicated, induction time was significantly shortened, and also adverse reactions such as excitement or incontinence were less frequently observed. Furthermore, isoflurane anaesthesia with MB premedication showed high respiratory rate and SPO2 value, compared with that of isoflurane monoanaesthesia. In conclusion, premedication with MB is effective for the attenuation of respiratory depression induced by isoflurane anaesthesia in mice, with rapid induction and less clinical adverse reaction.

1P019-S Effect of combination anesthetic (Medetomidine/ Midazolam/ Butorphanol) in mouse embryo transfer

○Rieko Takanashi,Motohito Goto,Yukiko Shimizu,Tadashi Okamura

Department of Laboratory Animals, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan

The mouse embryo transfer is a common operative procedure in reproductive engineering technology. Avertin(A) has been the standard anesthetic in mouse transgenic work. However, A can cause peritonitis in mice. Pentobarbital(P) is also widely used for injectable anesthesia in animal experiment. Disadvantages of P include a poor analgesic activity and narrow margin of safety. Recently, it has been reported that a combination anesthetic (M/M/B; Medetomidine/ Midazolam/ Butorphanol) is useful for laboratory rodents anesthesia. In this study, we evaluated the effects of the following anesthetics as surgical anesthesia in mouse embryo transfer. Groups of pseudopregnant ICR mice were weighed and injected with M/M/B(0.75/4/5mg/kg), A(tribromoethanol, 375mg/kg), P(50mg/kg) and administered 2% isoflurane(I). After anesthesia, fertilized eggs are surgically transferred to the oviducts of pseudopregnant mice. 44.6±14.7%, 42.3±15.0%, 54.9±3.4%, and 42.3±13.8% of transferred embryos were born from M/M/B, A, P and I anesthetic groups, respectively. There was no significant difference. Injectable drugs are a convenient way to administer anesthesia to rodents in biomedical research. The M/M/B anesthetized mice can be reversed with an alpha2-adrenoceptor antagonist, atipamezole. Therefore, we conclude that M/M/B is more suitable anesthetic for surgical operation in the mouse embryo transfer.

S 96 1P020-S Effect of anesthetics on salivary secretion in mice

○Masakatsu Nohara,Atsushi Tohei,Shiori Sasaki,Hiromi Amao

Laboratory of Experimental Animal Science, Nippon Veterinary and Life Science University, Tokyo, Japan

Objective: We aimed to compare the effect of three types of mixed anesthetic agents [medetomidine (Med), midazolam (Mid), and butorphanol (But)] with pentobarbital on salivary secretion in mice. Material and Methods: Colony-bred, mature male IQI mice were used throughout the experiment and all agents were injected intraperitoneally. Mice were treated with a mixture (Med/Mid/But: 0.3/6.0/7.5 mg/kg) or pentobarbital (40 mg/kg) and ten minutes later, they were additionally treated with pilocarpine (0.5 mg/kg) to enhance salivary secretion. Collection times of saliva were separated into two fractions (0-20, and 20-40 min). After the end of saliva sampling, atipamezole (0.3 mg/kg) were injected into the mice treated with the mixed agents. The levels of protein and corticosterone were measured by the fluorescence or the enzyme immunoassay (EIA), respectively. Results: The volume of saliva secreted in earlier time period was significantly higher than in later period in both anesthetic groups (P < 0.05), but the levels of protein and corticosterone were not significant difference between two fractions. The difference of two types of anesthetic did not affect the volume of saliva. The injection of pentobarbital reduced the body weight in mice, while the injection of mixed agents did not. Conclusion: The mixed anesthetic agents are recommended agent for saliva sampling, and the collected saliva was available for EIA of corticosterone.

1P021-S Bronchoalveolar and pleural lavage fluid cytology for lung toxicity assessment in rats

○Hiroshi Takehara,Maki Makita,Yasufumi Ota,Noriyo Natsume, Yukari Odagiri, Ryouta Tanaka,Mai Tsuchiya,Masato Naya,Makoto Hayashi Public Interest Incorporated Foundation BioSafety Research Center, Shizuoka, japan

Usefulness of bronchoalveolar lavage fluid (BALF) and pleural cavity lavage fluid (PLF) as an experimental material was evaluated for the assessment of pulmonary toxicity of chemicals in rats. There was no essential difference between diethyl ether or isoflurane for euthanasia/anesthesia, therefore isoflurane can be used for the purpose without any problems. Here, we also recognized phosphate buffered saline (PBS) and distilled water (DW) equally as a solvent/vehicle for negative control. PLF is also provided as a useful target material as well as BALF for assessing chemical lung toxicity. We may conclude that the intratracheal treatment and use of BALF and PLF as a target material is a good method for assessment chemical lung toxicity in rats. To evaluate the method, we used zinc chloride as a model chemical and obtained the expected and satisfied results.

S 97 1P022-S Endotracheal intubation to rats using an endoscope system 1 1 2 3 4 ○Kenjiro Konno , Naoki Itano ,Teppei Ogawa , Mika Hatakeyama , Kyoko Shioya , Noriyuki Kasai5 1Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, Japan 2NATSUME SEISAKUSHO CO., LTD., Tokyo, Japan 3AVS Co., Ltd, Tokyo, Japan 4Laboratory of Animal Experiment and Medicine Management, National Cerebral and Cardiovas-cular Center, Suita, Japan 5Center for Laboratory of Animal Research, Tohoku University, Seidai, Japan

Appropriate and effective anesthesia is critical because it has a strong influence on laboratory animals, and its affect greatly impacts the experimental data. Inhalational anesthesia by endotracheal intubation is currently prevailing in general anesthesia and is prefered over injection anesthesia. However it is not common for small laboratory animals. Therefore we assessed the capability of use for mice of the endotracheal intubation by using the endoscope system "TESALA AE-C1" and inhalational anesthesia using a ventilator. The intubation was successfully performed on all 12 Wistar rats (body weight: 178 - 265 g) intraperitoneally injected with M/M/B: 0.3/4/5 comprised of medetomidine, midazoram and butorphanol, at a dose of 0.3 mg/kg + 4.0 mg/kg + 5.0 mg/kg body weight/rat, respectively. After the intubated rats were connected with the inhalational anesthesia circuit, they were intraperitoneally injected with atipamezole (1.5 mg/kg b.w.). Vital signs were measured until 15 minutes after the connection. The data showed stable and normal values, which indicated that this new endotracheal intubation method was simple, reliable, and safe for rats, which means that this anesthesia is favorable in regard to the animal's welfare.

1P023-S Effects of Different Euthanasia Methods on Hematological Parameters in Rats and Mice 1 1 2 1 ○Misao Terada ,Hiroe Kon ,Toshio Akimoto ,Motoo Shinoda

1Laboratory Animal Research Center, Dokkyo Medical University, Tochigi, Japan 2Division of Laboratory Animal Science, Nippon Medical School, Tokyo, Japan

In animal experiments, hematological tests are commonly-performed. Small experimental animals are euthanized in case of whole blood collection. In the present study, we examined the effects of different euthanasia on hematological parameters in rats and mice. We divided Wistar male rats and BALB/c male mice into three groups, namely 1) cervical dislocation group, 2) isoflurane inhalation group and 3) diethyl ether inhalation group. After euthanasia, blood samples were collected through the heart for complete blood count and analysis of serum chemistry with Vet Scan HM2 and Vet Scan VS2. 1) In cervical dislocation group of rats, HCT, PLT and AMY were higher, and MCV was lower than the other groups. In mice, ALT was higher, and HGB, HCT, MCH, MCHC, PLT, ALP and T-Bil were lower than the other groups. 2) In isoflurane inhalation group of rats, T-Bil and Glu were higher, and ALB, ALP, BUN, Na+ and TP were lower than in the other groups. In mice group, BUN was higher, and ALB and Na+ were lower than the other groups. 3) In diethyl ether inhalation group, Glob was lower than the other groups in rats, and Glu was higher than the other groups in mice.Present study indicates that there are some differences of hematological parameters between each euthanasia methods, and these results suggest the necessity of considering selection of the euthanasia methods.

S 98 1P024-T Investigation of carbon dioxide euthanasia chamber for the mouse using commercial cage

○Eikichi Kamimura, Noriyoshi Hashimoto, Yohei Aoko, Yukiko Nakamura, Masahide Asano Institute for Experimental Animals, Kanazawa University Advanced Science Research Center, Kanazawa, Japan

We have devised a euthanasia apparatus for mice with a commercially available cage. A tube from the carbon dioxide gas supply was connected to a metal inlet adapter mounted on center top of the lid which was covered by transparent plastic sheet. When carbon dioxide was set at a flow rate of 1 L per minute, the mice lost consciousness within 3 minutes and were euthanized. This apparatus was prepared at low cost and seemed to provide an economically beneficial method for euthanasia of the mice.

1P025-S A case report of treatment in a cynomolgus monkey which showed immune-mediated hemolytic anemia

○Hiroshi Manabe,Mitsuhiro Shinomiya ,Yoshie Manabe,Tomio Hirota, Kenji Moriyama, Fumio Morita Toxicology Laboratory, Tokushima Research Center, TAIHO PHARMACEUTICAL CO., LTD.

A male cynomolgus monkey showed anemia, increases in reticulocyte counts and platelet counts in February, 2008. The animal was treated iron medicine (hereafter, IM) and vitamin B12 (hereafter, VB12) for hypoferric anemia for three months from March, 2008. The treatment was discontinued because hypoferric anemia was recovered by the therapy. However, the animal showed aggravation of general conditions again from September, 2008, three months later after the therapy cessation. Then severe anemia, increase in globulin and abnormal erythrocyte morphology were revealed by the clinical pathology in February, 2009. This animal was made a diagnosis of IMHA based on the above examinations. IMHA symptom was improved by the combination regimen of prednisolone (hereafter, PSL), IM and VB12 from March, 2009. In the treatment, we replaced PSL with adrenocorticotropin preparation of artificial composition (hereafter, ACTH) due to the dosing period of the PSL passed long-standing from January, 2010. In the result, we found that the regimen of 2-days on / 5-days off for ACTH and 3-days on / 4-days off for IM was optimal. In conclusion, it would be recommended for IMHA in monkey as a combination regimen that PSL, IM and VB12 were effective for the acute phase and then the maintenance therapy with ACTH and IM after symptom improvement was successful.

S 99 1P026-T Effect of frequent blood sampling in common marmosets 1 1 2 2 ○Nozomi Nakano , Yuko Katakai ,Tadahiro Sasaki ,Tsuyoshi Kurosu ,Kazuyoshi Ikuta2and Yasuhiro Yasutomi3 1The Corporation for Production and Research of Laboratory Primates 2Research Institute for Microbial Diseases, Osaka University 3Tsukuba Primate Research Center, National Institute of Biomedical Innovation

Under ketamine anesthesia blood samples (1mL) were collected from common marmosets on day 0, 1, 2, 3, 4, 5, and 7 and analyses in hematology and serum biochemistry were performed. As no lethal conditions nor severe health problems were not observed in the course of the experiment, collection of blood every day seemed possible. Hematological abnormalities suggested the propensity for anemia and requirement for the modification of collection volume and interval, and animal healthcare.

1P027-S Developing of Footprint Analysis System Using Infrared Depth Sensor

1 3 2 ○Akihiro Nakamura ,Hiroyuki Funaya ,Tomohiro Suzuki ,Shigeharu Wakana2,Tomohiro Shibata1,3 1Graduate School of Information Science, NAIST, Nara, Japan 2RIKEN BioResource Center, Tsukuba, Japan 3LSSE, Kyutech, Kitakyushu, Japan

Introduction: Most of the current footprint analysis systems make the subjects walk on transparent floors or treadmills, this can usually affect the subject's behavior.In our study, we developed a footprint analysis system that affects the subject's less compared to other current existing systems. Methods: Our system measures the subject's behavior in an open-field setting from underneath using an infrared depth sensor. The floor of the open-field was covered with tiled infrared-pass filters. When the subject was walking, the pixels obtained from the subject's infrared image that had top 3% luminance and was continuously stable in frames were regarded as the footprint. The first of our experiments investigated the behavioral difference of the mice in transparent and opaque floor conditions. The second experiment measured the gait of mutant mice and wild type mice. Result and Discussion: In the first experiment, the subjects showed difference in activity and time spent in the center area. This difference suggests that floor transparency affects the animal's behavior. In the second experiment, the gaits showed significant differences in stride length, swing time and stand time between mutant mice (D2;B6-Rgsc651/Rbrc) and wild type mice. Future Works: Presently, the system has been used only on black mice. Extension to other various colored mice is a possible future work of our study.

S 100 1P028-S A simple method for microinjection into the newborn mouse brain to study cerebellar developent 1 2 ○Makoto MIYAGAWA ,Hiroyuki ABE 1Lab. Anim. Cent., Teikyo Univ., Tokyo, Japan 2Dept. Judo Therapy, Facult. Med. Technol., Teikyo Univ., Utsunomiya, Japan

Objectives Cerebellum develops through dynamic morphological changes after birth. The granule cell layers changes a lot, where fundamental cellular events were involved. To study these events, this work aims at establishment of a simple method to inject the cerebellum of newborn mice with bioactive reagents. Materials and MethodsICR mice (P1-P4) were anesthetized by induced hypothermia, and were gently injected with some markers using a 30G needle. First, we injected brain hemisphere. The position of the injection site on the skin relative to that of bregma was determined using a caliper. The pups were then marked, recovered in both palms and returned to the litter. They were sacrificed at days 1, 7 and 21 later, and the brains were analyzed by histological ways. ResultsWe did not see any accidental death of the pups from the manipulation itself. The increase in both body and brain weights was not affected by the manipulation. 3ul of 1% Evans Blue (EB) in DPBS was successfully injected. Histological analyses showed that this dye resides in the injected sites at 1 day but not 1 week after injection. DiscussionThis method is effective for intrabrain injection of as much as 3ul of reagents. Since EB left the injected sites in 1 week, we are testing larger MW markers and lipophilic markers. Exploration of the injected sites for the presence of reactive gliosis is proceeding. Finally, we are now injecting cerebellum.

1P029-S Comparison of multiple blood sampling methods in mouse and rat

○Kiyoshi Morii,Hiroshi Tsujii,Akio Kihara,Michiko Hashimoto,Hironari Koyama

Astellas Research Technologies Co. Ltd., Laboratory Animal Science Div., Tsukuba, Japan

The most appropriate blood sampling method should be selected depends on the study objects and conditions (i.e. species, strains, clinical conditions, quality/quantity of blood required, frequency/interval/speed of sampling, technical efficiency, and anesthetics). However, sampling methods written in published texts would be occasionally inapplicable due to the experimental condition, characteristics of animals and technical problem. In addition, replacement of retro-orbital plexus route has been an issue. We give investigators guidance about following methods.Based on the experience, we introduce availability of three sampling routes in mice and rats. Any samplings were conducted within the allowable range of published guides.1)Caudal vein sampling with using butterfly needle minimizes coagulation, hemolysis, and contamination of tissue fluid and is recommended for repeated (>10 times in rat) on-time sampling without anesthesia. Not applicable for over 0.1mL sampling or under five weeks of age in mice. 2)Jugular vein is recommended for high quality sampling without coagulation, hemolysis, and contamination. Around 0.1mL is obtainable in mice without anesthesia and 0.5mL in rats. Multiple sampling is possible. 3)Submandibular vein is useful for rapid and certain amount of sampling (approx. 0.1mL without anesthesia in mice).These three routes are applicable for most of the studies which requires multiple sampling by providing sufficient training.

S 101 1P030-T Application of the endoscope for small animals in beagle dogs

○Masaki Nakayama, Isamu Nakatsu, Tadayoshi Takemoto, Hirohiko Goto

Otsuka Pharrmaceutical Co., Ltd, Department of Toxicology, Tokushima, Japan

[POURPOSE] Application of endoscope was examined in anesthetized or conscious dogs. [METHODS] 1. Endoscope for small animals (VQ-5112B, OLYMPUS) was used for examination of the duodenum and collection of gastric fluid using anesthetized beagle dogs. 2. Ileus tube with double-balloon was also used under anesthesia. 3. Endoscopy of the stomach and duodenum also examined in conscious dogs. [RESULTS AND DISCUSSION] In anesthetized dogs, examination of the stomach and collection of gastric fluid were easily conducted. Gastric fluid was collected via an endoscope or via a gastric tube which was guided with grasping forceps through the endoscope. Endoscope insertion into the duodenum depended on the pylorus condition. The ileus tube was inserted into the stomach, and the tube was guided into the duodenum by straight grasping forceps. The balloons extended before and behind the duodenal papilla, and washed the inside with saline and collected the fluid. In case of using acclimated dogs to a training of swallowing flexible catheter, intubation of the endoscope was possible under awaking condition with lateral position. In conscious dogs, insertion of endoscope to the duodenum was difficult due to the active movement of the peristalsis, but gastric fluid was easily collected.

1P031-T An improved surgical technique increases the survival rate of lung cancer SOI model

○Chisako Ishimaru, Ryo Nakamura, Toshihiko Shiga, Hitoshi Arakawa, Makoto Monnai

Kamakura Branch, Chugai Research Institute for Medical Science, Inc.

Orthotopic tumor models give better representations of the onset of metastasis and are more suitable for testing the efficacy of various anti-cancer agents than subcutaneous tumor models. However, surgical orthotopic implantation (SOI) models of lung cancer are reported to have high perioperative mortality. In this study we aimed to improve the survival rate after SOI via thoracotomy. At first, we decided the control settings of an appropriate ventilator, and improved a 22G i.v. catheter so as not to damage the trachea. The mouse was anesthetized by isoflurane inhalation (flow rate: 3.8L/min) using a ventilator for small animals (respiration rate: 150 strokes/min, flow rate: 0.30~0.40L/min) and intubated using the improved 22G i.v. catheter. Using these settings and devices, we implanted a 1-mm3 piece of xenograft tumor into the left lung of the SCID mouse via thoracotomy. The survival rate after implanting was 100% and tumor formation at the implantation site was observed in all mice after 14 days. This suggested that a procedure that uses a ventilator for small animals and our improved 22G i.v. catheter can maintain a high probability of survival in the SOI model of lung cancer.

S 102 1P032-T An attempt at training cynomolgus monkeys to have affinity for humans

○Ai. Nishimoto, Kanako. Fujiwara, Koru. Takaura, Yuki. Tachibana, Yuji. Sudo, Yasuhiro. Sakurai, Hironari. Koyama Laboratory Animal Science Div. Astellas Research Technologies Co., Ltd.

[Objective] We attempt to train monkeys in having blood collected or receiving drugs in an unrestrained condition as a goal. First, they were trained to have an affinity for personnel.[Method] Monkeys aged between 3 and 12 years received raisins more frequently (once to three times a day) and had more communication with personnel (three to five times a day) for 4 months. The behavior was scored as following; −1: frightened of humans; 0: does not eat in the presence of humans; 1: picks up raisins in the presence of humans when served in the cage; 2: receives raisins by hand through the upper part of the cage; 3: receive raisins by the lips through the upper part of the cage; 4: receives raisins by hand through the lower part of the cage; 5: receives raisins by the lips through the lower part of the cage.[Results] After the training, the number of animals that could not receive raisins directly from personnel decreased about 80%, and the mean score increased from 1.1 to 4.5. The score began to improve after 12 days training, and stereotypic behavior or fecal smearing activity improved in some animals. These results showed that the affinity of monkeys for humans markedly increased through more frequent feeding and communication.

1P033-T Social Housing for Macaques

○Hiroyuki Satake, Shigetoshi Takemoto, Naganuma, Taishiro Kimura, Kiyoshi Morii, Michiko Hashimoto, Hironari Koyama Astellas Research Technologies Co., Ltd., Ibaraki, Japan.

Social Housing for MacaquesSatake H, Takemoto S, Naganuma Y, Kimura T, Morii K, Hashimoto M, Koyama HAstellas Research Technologies Co., LtdLike all social animals, nonhuman primates should normally be kept in social housing (The Guide for the Care and Use of Laboratory Animals; 8th Edition). Since it is reportedly difficult to start social housing in mature macaques that has been housed singly, a suggestion has been made to start social housing for macaques at a young age. Here we report about a trial for social housing (pair or group housing) in aged monkeys.The macaques (cynomolgus and rhesus; 3-16 years old) were engaged in trial since Jan 2012 to Dec 2013. Cages for group housing or connectable cages for individual housing were used.We implemented social housing with a high success rate (95.5%, as of Dec 31st, 2013). Even in elderly macaques that were over 10 years old the success rate was at 87% (100% in cynomolgus, 60% in rhesus). Group housing resulted in improvement of stereotypic behavior or hair loss in some animals. However, 33 incidents of biting during fighting have been observed in 2years, and some concerns such as feed intake differences exist.The assessment of compatibility of individual macaques is the most important factor in making pairs or groups of mature macaques. Bites can be treated with sutures. Housing animals individually only during feeding time is effective when there are feed intake differences.

S 103 1P034-T Effect of light-dark stress on tumor metastasis by using mouse transplantable syngeneic tumor 1 1 1 2 ○Katsuko Naitou , Akemi Kamijo , Kayoko Iwao , Kyoji Ogoshi

1Support Center for Medical Research and Education, Tokai University 2Hidaka Hospital, Japan

Effect of light-dark stress on tumor metastasis by using mouse transplantable syngeneic tumor.Katsuko Naitou, Kayoko Iwao, Akemi Kamijo 1) Kyoji Ogoshi 2) 1)Education and Research Support Center (Isehara),Tokai University 2)Hidaka HospitalIntroductionSeveral stressors may effect on the human physiology. ((Introduction))Several stressors may effect on the human physiology. We evaluate the effect of light-dark stress on tumor metastasis by using mouse transplantable syngeneic tumor.((Materials and Methods)).1.LLC(Lewis Lung Carcinoma), transplantable tumors, of 7X103 cells, were used and injected into the muscle of the right leg. Body weight, tumor weight, number of metastasis and metastatic area in lung were evaluated after 18 days tumor injection.2.Light-dark stress. A group: Light 12 hours, dark 12hours (standard animal care environment). B group: Light 24 hours 15 days.C group: Light 24 hours 7 days.D group: Dark 24 hours 15 days.((Results))(1)Body weights of B, C, D group increased in the order of C, D, B more than A group.(2)Tumor weight increased in D group.(3)Number of metastasis in lung showed lower in C group.(4)Metastatic area in lung showed lower in B group (p=0.059).((Conclusion))These findings suggest that light (24 hour, 15 days) stress may antitumor effect on tumor metastasis. We will further evaluate the effect of antitumor substance on this stress condition.

1P035-T Analysis of X-ray CT data of mice using a freely downloadable image analysis software 1,2 2 1 1 ○Kumiko Gotoh ,Masako Shimamoto ,Yoshioki Shiraishi ,Tatsuya Shimasaki ,Seiji Okada3,Akihiro Kojima1 1Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan 2 Graduate school of Medical Sciences,Kumamoto University, Kumamoto, Japan 3 Center for AIDS Research, Kumamoto University, Kumamoto, Japan

By using a computer image processing free software, it is possible to analyze and process X-ray CT data of the small animal molecular imaging system easily and inexpensively. In our technical assistance for the evaluation of reduced bone density and fat measurement of mice, we realized that this free software provides enough accuracy, and was easier to use than the imaging system dedicated software.

S 104 1P036-T Study of Cherenkov luminescence imaging for small living animals with Iodine-131

1 2 2 1,2 ○Masako Shimamoto , Kumiko Goto , Yoshioki Shiraishi , Tatsuya Shimasaki , Akihiro Kojima1,2 1Division of Radioisotope Science, Graduate School of Medical Sciences, Kumamoto University, Kumamoto Japan 2 Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan

By detecting fluorescence and luminescence in small animals, real-time in vivo optical imaging systems build images of various biological and pathological conditions. The beta rays emitted from the radionuclide can be detected as Cherenkov light and used in image construction. In this study, we report the result of a basic study of Cherenkov light imaging of radioactive iodine (I-131).

1P037-S Anesthetic effects of a three-drugs mixture and antagonistic effects of atipamezole in mice 1 1 1 2 3 ○Yumiko Kirihara ,Mayumi Takechi ,Kaoru Kurosaki ,Yoji Saito ,Yuta Kobayashi

1Department of Experimental Animals, Interdisciplinary Center for Research, Shimane University, Izumo, Shimane, Japan 2Department of Anesthesiology, Faculty of Medicine, Shimane University, Izumo, Shimane, Japan 3Department of Fundamental Nursing, Faculty of Medicine, Shimane University, Izumo, Shimane, Japan

Objective: The anesthetic mixture of medetomidine (Med), midazolam (Mid), and butorphanol (But) has been used by intraperitoneal (ip) injection in mice. However, other administrative routes may cause different anesthetic effects. Then, we examined anesthetic effects of the anesthetic mixture by subcutaneous (sc) and intravenous (iv) compared to ip injection. Atipamezole (At) is an antagonist of Med. We examined how different doses, routes and injection timing of At affect the recovery from anesthesia. Methods: We used ICR mice. In experiment 1, we injected the anesthetic mixture by three routes and measured anesthetic scores. In experiment 2, we administered 0.3 and 1.5 mg/kg of At by ip and sc routes at 10 and 30 min after injection of the anesthetic mixture and then measured anesthetic scores. Results and Discussion: There were no significant differences of anesthetic duration among the three injective methods. Antagonistic effects of At 0.3 and 1.5 mg/kg by ip injection worked equally when administered at 30 min after the anesthetic mixture. The antagonistic effect of At 1.5 mg/kg was strongest at 10 min after the anesthetic mixture. These results may contribute to the welfare of laboratory animals.

S 105 1P038-S MafB promotes atherosclerosis by inhibiting foam-cell apoptosis 1 1 1 1 2 ○Michito Hamada ,Megumi Nakamura ,Mai Tran ,Dinh Tra ,Satoko Arai ,Cynthia Hong3,Toru Miyazaki2,Peter Tontonoz3,Hitoshi Shimano4,Satoru Takahashi1 1Department of Anatomy and Embryology Faculty of Medicine University of Tsukuba 2Division of Molecular Biomedicine for Pathogenesis University of Tokyo 3Department of Pathology and Laboratory Medicine, Howard Hughes Medical Institute, University of California 4Department of Internal Medicine University of Tsukuba

MafB is a transcription factor that induces myelomonocytic differentiation. However, the precise role of MafB in the pathogenic function of macrophages has never been clarified. Here we demonstrate that MafB promotes hyperlipidemic atherosclerosis by suppressing foam-cell apoptosis. Our data show that MafB is predominantly expressed in foam cells found within atherosclerotic lesions, where MafB mediates the oxidized LDL-activated LXR/RXR-induced expression of apoptosis inhibitor of macrophages (AIM). In the absence of MafB, activated LXR/RXR fails to induce the expression of AIM, a protein that is normally responsible for protecting macrophages from apoptosis; thus, Mafb-deficient macrophages are prone to apoptosis. Haematopoietic reconstitution with Mafb-deficient fetal liver cells in recipient LDL receptor-deficient hyperlipidemic mice revealed accelerated foam-cell apoptosis, which subsequently led to the attenuation of the early atherogenic lesion. These findings represent the first evidence that the macrophage-affiliated MafB transcription factor participates in the acceleration of atherogenesis.

1P039-S Dendropanax morbifera extract ameliorates heavy metal-induced increased oxidative stress 1 1 1 1 ○In Koo Hwang ,Woosuk Kim ,Hyo Young Jung ,Dae Young Yoo ,Sung Min Nam1,Jong Whi Kim1,Jung Hoon Choi2,Ki-Yeon Yoo3,Moo-Ho Won4,Yeo Sung Yoon1 1Department of Anatomy and Cell Biology, College of Veterinary Medicine, and Research Institute for Veterinary Science, Seoul National University, Seoul, South Korea 2Department of Anatomy, College of Veterinary Medicine, Kangwon National University, Chuncheon, South Korea 3Department of Oral Anatomy, College of Dentistry, Research Institute of Oral Sciences, Gangneung-Wonju National University, Gangneung, South Korea 4Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon, South Korea

Dendropanax morbifera is used in traditional medicine and has antioxidant effect. However, there were few reports on the effects of Dendropanax morbifera on heavy metal induced brain damage. In the present study, we investigated the effects of D. morbifera extract on heavy metal-induced lipid peroxidation and antioxidants by enzyme-linked immunosorbent assay methods. The administration of mercury, lead, or cadmium significantly increased the lipid peroxidation judged from malondialdehyde (MDA) in hippocampal homogenates. In addition, administration of these heavy metals significantly reduced the levels of superoxide dismutase (SOD) and catalase (CAT). Oral supplements of Dendropanax morbifera extract significantly ameliorates the heavy metal-induced changes in MDA, SOD, and CAT. These results suggest that D. morbifera extract play a great role on heavy metal-induced brain damage by reducing lipid peroxidation and increasing antioxidants.

S 106 1P040-S Amelioration in myelin lesions by iron deficient diet in the dmy myelin mutant rat

1 1 1 1 ○Mitsuru Kuwamura ,Yuko Hasegawa ,Miyuu Tanaka ,Takeshi Izawa ,Jyoji Yamate1,Takashi Kuramoto2 1Laboratory of Veterinary Pathology, Osaka Prefecture University, Osaka, Japan 2Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University

The dmy rat is characterized by progressive myelin breakdown in the CNS. The homozygous dmy/dmy rats show ataxia from 5-6 weeks of age and die around 7-8 weeks. We demonstrated an abnormal iron accumulation in the CNS of dmy rats. To elucidate the roles of iron in myelin breakdown, we investigated the myelin lesions in the spinal cord of the dmy rats fed by iron deficient diet. Three homozygous dmy/dmy rats were fed by iron deficient diet (iron content 0%) and compared the lesion in the controls fed by usual diet (iron content 0.02%). Spinal cords were removed and examined histopathologically and immunohistochemically. All rats fed by usual diet became recumbency around 7-8 weeks of age. However, three rats fed by iron deficient diet were alive at 8 weeks. Usual diet-provided 8-week-old rats had severe myelin breakdown (vacuolation) in the ventral and lateral funiculi of spinal cords. In contrast, mild myelin lesions were found in the homozygous rats fed by iron deficient diet. In addition, mild glial responses were noted in the iron-deficient group, whereas prominent glial responses (activated astrocyte and microglia) were observed in the severely affected myelin lesions. These findings indicated that iron-mediated oxidative stress plays important roles for the progression of myelin breakdown in the dmy rats.

1P041-S Efficacy of histamine treatment for hydrodynamics-based transfection

1 1 2 1 ○Masahiko Fujisawa ,Yui Osaka ,Motoyo Maruyama ,Yoji Hakamata

1School of Veterinary Nursing and Technology, Nippon Veterinary and Life Science University, Tokyo, Japan 2Division of Laboratory Animal Science, Nippon Medical School, Tokyo, Japan

Improving the in vivo gene delivery method is an important topic for research. In this study, we examined the utility of the hydrodynamics-based transfection (HBT) method, which was performed using a rapid intravenous injection of a large quantity of solution (4-8 %/BW) containing naked DNA in 10 seconds. We should consider safety with regard to the circulatory system and target organ. We focused on enhancing the permeability effect of histamine (HIS), and attempted to evaluate the safety and efficacy of HIS treatment for HBT. We examined the effect of HIS during HBT (injected saline through the penile vein) in the liver. We examined the following experiments; 1) histological analysis, 2) permeability change, 3) gene transfer efficiency. Histological analysis showed that the central vein and sinusoid areas and the number of subcellular vesicles dose-dependently increased with an increase in the amount of HBT solution. Permeability of the liver dose-dependently increased with an increase in the histamine level. HBT solution with a low HIS dose (0.01 mg/kg) significantly increased the permeability of the liver. Exogenous luciferase gene expression increased in the HIS-treated group (0.01 mg/kg) , and its expression period prolonged. HIS pretreatment appeared to contribute to an increase in gene transfer efficiency by transient enhancement of permeability during HBT.

S 107 1P042-S Research on dental caries in Zucker rat strains

1 1 2 2 ○Yasushi Kodama ,Taiki Nishimoto ,Tomoya Sano ,Kiyokazu Ozaki ,Yoshihiko Taniguchi1,Tetsuro Matsuura2 1Hiroshima International University, Kure, Japan 2Setsunan University, Hirakata, Japan

It remains unclear how obesity and diabetes influence caries development in Zucker fatty rats (ZF) and Zucker diabetic fatty rat (ZDF). Furthermore, diabetes is related closely to dysfunctional salivary secretion in human. The aim of this study was to clarify the effect of obesity and diabetes on caries development, and the relationship between salivary gland and caries development in Zucker strains. Male ZDF, ZF, and Zucker lean rats (Lean) as control were used. Body weight (BW) and blood levels of triglyceride (TG), total cholesterol (T-Cho) and glucose (BG) were measured. All rats were sacrificed at 43 weeks of age for histopathological examination. The ZF' average BW was more than twice that of the other 2 groups, but the ZDF and Lean had comparable BW. The average TG and T-Cho levels were elevated in the ZF and ZDF, but the ZF' average TG and T-Cho levels were three times and twice, respectively, those of the ZDF. The ZDF demonstrated a high BG level. The ZF had a significantly higher BG level than that of Lean but had about one-half that of the ZDF. Histopathologically, vacuolization of acinar cells was observed in the parotid and lingual glands of ZDF and ZF; the incident and frequency of these lesions in ZDF were higher than ZF. Dyslipidemia and hyperglycemia synergistically enhance latent caries in Zucker strain rats; hyperglycemia enhances caries more than dyslipidemia. Dental caries may result from dysfunctional salivary gland.

1P043-S Relationship between disorder of salivary glands and dental caries in type 2 diabetic mice 1 1 2 2 ○Yuka Mitsui ,Kiyokazu Ozaki ,Taiki Nishimoto ,Yasushi Kodama ,Yoshihiko Taniguchi2,Tetsuro Matsuura1 1Setsunan University, Hirakata, Japan 2Hiroshima International University, Kure, Japan

Background: Dental caries are produced in a model of type 2 diabetes, db/db mice. Meanwhile, hyperglycemia causes the abnormal function of salivary glands in human. In this study, we sought to investigate functional and morphological changes in salivary glands of diabetic db/db mice.Methods: Male and female db/db mice aged 40 weeks along with age-matched db/+ mice were used. The saliva volume was measured by stimulation of pilocarpine at 40 weeks of age. The serial sections of defatted salivary glands in other mice without stimulation of pilocarpine were subjected to histopathological examination. The mandibular and maxillary molars were examined by macroscopy for evaluating dental caries. Results: The incidence and severity of molar caries in db/db strain were much higher than in db/+ strain. The average saliva volume in db/db mice was remarkably decreased and was less than half that of db/+ mice. Morphologically, it was impossible to find salivary glands embedded in adipose tissue in db/db mice. Lobular atrophy of the parotid glands was observed in large numbers of db/db mice, and the incidence was significantly higher than that of db/+ mice in both sexes. However, acinar atrophy was slightly increased only in male db/db mice. Conclusion: The onset of caries is related to depressed salivary function in db/db mice, and yet the progression of morphological salivary change might be rather mild.

S 108 1P044-S Dental Caries in Alloxan-induced Diabetic Mice

1 1 2 2 ○Teturo Matsuura ,Hayato Maruyama ,Taiki Nishimoto ,Yasushi Kodama ,Yoshihiko Taniguchi2,Kiyokazu Ozaki1 1Setsunan University, Hirakata, Japan 2Hiroshima International University, Kure, Japan

Background: Alloxan-induced hyperglycemia causes rapid-onset and progressive dental caries in rats. However, these rats are considerably excluded from the experimental study because of ketoacidosis and urinary tract infection. In this study, we evaluated the AL-induced diabetic mouse as a dental caries model using ICR strain widely used for experimental animals. Methods: Diabetic conditions were induced in male ICR mice by single intravenous injection with 75 mg/kg of AL (DM). Age-matched untreated mice were used as control. Mice were sacrificed at 1 and 3 months after AL treatment, and all molars were morphologically examined for evaluating dental caries. Results: Severe hyperglycemia persisted during experimental period in DM group. The ratio of death due to AL treatment was merely 5%, and no diabetic animals returned to normoglycemia. Body weight increase was slightly but significantly suppressed compared to control group (p<0.05). The average food and water consumption were 2.5 times and 7 times, respectively, those of the control group. The partial coronal defect was detected in a few cases of DM group, and the incidence tended to increase with age. Conclusion: AL injection induced severe long-lasting hyperglycemia in almost ICR mice and caused milder dental caries compared to AL-treated rats. AL-induced diabetic ICR mice are considered to be useful model for diabetic dental caries.

1P045-S Effects of high-fat diet on mammary tissue physiology and subsequent carcinogenesis in rats 1 1 2 2 1 ○Toshio Imai ,Naoaki Uchiya ,Gen Fujii ,Michihiro Mutoh ,Mami Takahashi

1Central Anim. Div., Natl. Cancer Ctr. Res. Inst, Tokyo, Japan 2Div. Cancer Prev. Res., Natl. Cancer Ctr. Res. Inst, Tokyo, Japan

[Background] Changes into Westernized lifestyles should increase the risk of breast cancer. Asian immigrants to US were reported to show adipokine dysregulation. [Aim] To clarify the effects of high-fat diet (HFD) on mammary tissue physiology and subsequent carcinogenesis in rats. [Methods] Female F344 rats at 5-6 weeks of age were fed a basal, 10% corn oil or a commercially available beef tallow diets for 3 days - 5 weeks, and they were euthanized followed by sampling of blood and mammary gland. In addition to similar feeding of HFD for 5 weeks, other females were gavaged DMBA and euthanized at 33 weeks of age. [Results] Serum leptin levels were elevated in the HFD groups as compared to the basal diet (BD) group. Phospho-STAT3:STAT3 ratio in mammary tissues was higher in the HFD groups than that in the BD group, indicating activation of leptin signaling. DNA microarray and qPCR analyses revealed elevation of cell adhesion molecule-A (CAM-A) gene expression in mammary tissue of rats fed HFD. For carcinogenesis, incidence, multiplicity and volume of mammary carcinomas were increased by HFD. In comparison of CAM-A gene expressions in carcinoma/normal mammary tissue, its elevation was found in some rats fed HFD. [Conclusion] HFD is likely to be associated with tumor promotion via hyperleptinemia with activation of STAT3 signaling and elevation of CAM-A gene expression in the mammary gland.

S 109 1P046-S Canine Secondary Pulmonary Hypertension: considered as a disease model 1 2 1 1 ○Takao Kanai ,Shuichi Chimura ,Hidehiro Ueshiba ,Yoshihiko Miyakawa ,kazunori Hirukawa1,Masafumi Yamanaka1,Mamoru Yoneyama1,Miho Koizumi1 1Institute of Laboratory Animals, Tokyo Women's Medical University, Tokyo, Japan 2Chimura Animal Hospital, Iwakura city, Japan

Pulmonary hypertension(PH)is observed while little to one of the human respiratory, cardiovascular disease similar.Latest diagnostic classification of PH is a Dana Point of modification in 2008. We observed a PDA + PH in dog.Miniature-dachsfund,5-month-olds,male.(Course)slightly poor growth, cardiac disease, suspected. After treated pulmonary edema and stable breathing shallow and fast. In auscultation, listening to the noise apex LV and left heart base. Both heart load pattern in the electrocardiogram Some pulmonary congestion and severe left ventricular dilatation in thoracic X-ray. In echocardiography, PDA,VSD, main PA expansion, and pulmonary valve regurgitation mild to severe LV volume overload findings, the AV under, diagnosed with VSD + PDA. PDA surgery performed due to the lack of the Eisenmenger. After surgery,the stable postoperative, and died of respiratory failure dyspnea occurs gradually, were necropsied. (Pathology)the apex consists of the RV, the right side of the heart spherical system is expanded. RV is lumen expansion, and there is wall thickening.PDA in extracardiac, VSD in intracardiac were found. Lung: PA had jumped to the in the lung cut surface. PD:VSD,PDA,PH.(Discussion)We report examined the possibility of human PH model based pathological findings(EBM) of PH lesions in dog were observed in human similarly.

1P047-S Expression of 5-HT1A receptor in astrocyte in the hippocampal CA1 region following cerebral ischemia 1 1 1 2 ○Joon Ha Park ,In Hye Kim ,Jeong-Hwi Cho ,Jung Hoon Choi ,In Koo Hwang3,Ki-Yeon Yoo4,Moo-Ho Won1 1Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon, South Korea 2Department of Anatomy, College of Veterinary Medicine, Kangwon National University, Chuncheon, South Korea 3Department of Anatomy and Cell Biology, College of Veterinary Medicine, Seoul National University, Seoul, South Korea 4Department of Oral Anatomy, College of Dentistry, Research Institute of Oral Sciences, Gangneung-Wonju National University, Gangneung, South Korea

5-hydroxytryptamine (5-HT, serotonin) plays protective or detrimental roles in the development of ischemic damage. In the present study, we investigated the time-course changes in 5-HT1A receptor protein expression in the gerbil hippocampal CA1 region after transient global cerebral ischemia. 5-HT1A receptor immunoreactivity in the stratum pyramidale (SP) of the hippocampal CA1 region was decreased from 6 h, and hardly observed at 1 day and 2 days after ischemic damage. At 5 days and 10 days after ischemia/reperfusion, 5-HT1A receptor immunoreactivity was increased and detected newly in astrocyte of the ischemic CA1 region. The pattern of changes in 5-HT1A receptor protein levels in the hippocampal CA1 region after I/R was similar to that observed in the immunohistochemical data. These results indicate that 5-HT1A receptor protein expressions may be related with the ischemia-induced neuronal death and the function of astrocyte in the ischemic hippocampal CA1 region.

S 110 1P048-S Changes and expressions of Id proteins in the gerbil hippocampus following global ischemia 1 1 1 2 ○Jeong-Hwi Cho ,Joon Ha Park ,In Hye Kim ,In Koo Hwang ,Jung Hoon Choi3,Ki-Yeon Yoo4,Moo-Ho Won1 1Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon, South Korea 2Department of Anatomy and Cell Biology, College of Veterinary Medicine, Seoul National University, Seoul, South Korea 3Department of Anatomy, College of Veterinary Medicine, Kangwon National University, Chuncheon, South Korea 4Department of Oral Anatomy, College of Dentistry, Gangneung-Wonju National University, Gangneung, South Korea

In the present study, we examined effects of ischemia-reperfusion injury on the immunoreactivity and protein levels of Id in the gerbil hippocampus following transient cerebral ischemia. The Id-1 immunoreactivity was detected in the nucleus of pyramidal neurons in the hippocampal CA1-3 regions, and maintained until 2 days post-ischemia. But, it was barely detected in pyramidal neurons from 5 days after ischemia-reperfusion; however, Id-1 immunoreactivity was detected in GABAergic interneurons of the ischemic CA1 region. Otherwise, Id-4 immunoreactivity was newly expressed in microglia, not astrocytes, in the ischemic CA1 region from 1 day post-ischemia. However, Id-4 immunoreaction was hardly detected in the ischemic CA3 region. Furthermore, Id-1 and Id-4 protein levels in the ischemic CA1 region were changed like the change pattern of its immunoreactivity. These results indicate that changes of immunoreactivity and protein levels of both Id-1 and Id-4 limited in the CA1 region may be related to the ischemia-induced delayed neuronal death.

1P049-S Silk fibroin hydrolysate ameliorates diabetic dyslipidemia in T2DM animal model ○HARRY JUNG, HAJIN NAM, JUN GYO SUH

Department of Medical Genetics, College of Medicine, Hallym University

The antidiabetic properties in silk materials, such as silk worm and silk fibroin, have been described in traditional folk medicine. In a previous study, we reported that the silk fibroin hydrolysate (SFH) had a protective effect against glucotoxicity in HIT-15 cells and an antidiabetic effect in a noninsulin-dependent diabetes mellitus (type 2 diabetes, T2DM) mouse model. This study aimed to determine whether SFH ameliorates diabetic dyslipidemia and promotes pancreatic β-cell function in a T2DM animal model (C57BL/KsJdb/db). SFH significantly improved blood glucose levels after 4 weeks of treatment. T2DM animals had a normal serum blood glucose level after 6 weeks of SFH treatment. Hemoglobin A1c (HbA1c) levels were significantly lower in T2DM animals that were treated with SFH for 6 weeks. Glucose tolerance in T2DM animals also significantly improved after 6 weeks of SFH treatment. Furthermore, plasma total cholesterol, high-density lipoprotein cholesterol, nonesterified fatty acid, and triglyceride concentrations significantly decreased in SFH-treated T2DM animals compared to T2DM animals. This result showed that SFH ameliorated diabetic dyslipidemia in the T2DM model mice. Taken together, our results suggest that SFH could be used as a potential agent to treat dyslipidemia in patients who have type 2 diabetes mellitus.

S 111 1P050-S Anti-obesity effects of a glucagon-like peptide-1 mimetic in diet-induced obesity mice

○Soraaki Takahashi,Sena Watanabe,Chika Arisato,Chihiro Kondo, Dai Nagakubo, Mitsuyuki Shirai,Fumitoshi asai Laboratory of Veterinary Pharmacology, School of Veterinary Medicine, Azabu University

Glucagon-like peptide-1 (GLP-1) mimetics are currently successfully used to treat patients with type 2 diabetes mellitus. This study examined the anti-obesity effects of the GLP-1 mimetic liraglutide in diet-induced obese mice. Male C57BL/6NCr mice were divided into 5 groups: the normal diet (ND) + saline, ND + liraglutide, high-fat diet (HFD) + saline, HFD + low doses of liraglutide (100 µg/kg), and HFD + high doses of liraglutide (200 µg/kg) groups. Saline or liraglutide was administered s.c. for 4 weeks beginning at 16 weeks of age. The body weight was significantly elevated in the HFD + saline group relative to that in the ND + saline group. In contrast, the increase in body weight was dose-dependently inhibited in the HFD + liraglutide group. No apparent difference in food intake was observed between the groups. The blood glucose levels were lower and blood insulin levels higher in the HFD + high doses of liraglutide group relative to those in the HFD + saline group. A dose-dependent decrease in the mesenteric fat weight was observed in the liraglutide-treated group. This study demonstrated that in diet-induced obese mice, the GLP-1 mimetic liraglutide exerted an anti-obesity effect. The fact that liraglutide administration did not lead to a decrease in food intake suggested that the anti-obesity effects of liraglutide might result from its enhancing effects on energy metabolism.

1P051-S Liver toxicity of 1,2-dichloropropane as inhaled in mice

○Rui-Sheng Wang,Megumi Suda,Yukie Yanagiba,Tetsuya Suzuki

Division of Health Effects Research, Japan National Institute of Occupational Safety and Health

1,2-Dichloropropane (1,2-DCP) is suspected as the causal substance of occupational cholangiocarcinoma. 1,2-DCP has been reported to induce acute liver damage at high concentrations in animals. It is concerned that whether it has any effect on liver at concentrations that may exist at workplaces. In this study, we examined the liver toxicity of 1,2-DCP with mice either after a single or repeated exposure. Male mice were exposed to 1,2-DCP at 300 ppm for 6 hr, and sacrificed at 4, 8 and 16 hr after termination of the exposure. In the repeated exposure experiment, mice were exposed to 1,2-DCP at 150, 300 and 600 ppm, 6 hr/day, 5 days/week, for 6 weeks, and sacrificed 18 hr after the last exposure. RESULTS: Degeneration of liver cells appeared from 8 hr in the exposed mice, and necrosis of centrilobular hepatocytes was evident at 16 hr. Plasma ALT and AST elevated from 4 hr. In the 6-week experiment, no obvious histological changes were found in mice of 150 and 300 ppm groups. Degeneration of hepatocytes was observed only in mice of 600 ppm group. Plasma ALT and AST were not elevated in any exposure groups. DISCUSSION: Acute liver damage was induced after a single exposure to 1,2-DCP at a concentration that might exist at workplaces. However, the liver damage became much mild following repeated exposure to 1,2-DCP, indicating that some kind of tolerance may occur to the tissue. Further studies are needed to elucidate the nature of hepatic damage induced by 1,2-DCP.

S 112 1P052-S The activities of wheel running and striatal dopaminergic neurons in adult male TSOD mice. 1 2 1 3 ○Atsushi Tohei ,Shu-ichi Kojima ,Hiromi Amao ,Yoshiyuki Sasaki ,Ryoji Hokao3,Motoo Shinoda4 1Laboratory of Experimental Animal Science, Nippon Veterinary and Life Science University, Tokyo, Japan 2Department of Pharmacology, Dokkyo Medical University, Tochigi, Japan 3Institute for Animal Reproduction, Ibaraki, Japan 4Laboratory Animal Research Center, Dokkyo Medical University, Tochigi, Japan

[Backgrounds & Aims] Tsumura, Suzuki, Obese Diabetes (TSOD) mice have reported to exhibit hyperphagia, hyperglycemia and hyperinsulinemia. We have previously examined the effects of pair-feeding on obesity in TSOD mice and reported that hyperphagia is not the sole cause of obesity in TSOD (Conference for Lab. Anim. Sci. and Tech. 2012). In the present study, we examined the activities of wheel running and striatal dopaminergic neurons in adult male TSOD mice. [Materials & Methods] Male TSOD and Tsumura, Suzuki, NonObesity (TSNO) as control mice (12 weeks of age) were used in the present study, and wheel running activity was monitored for 24h at 3 days after induction to wheel equipped cages. After behavioral analysis, mice were sacrificed by decapitation at 10:00 (dark period, 19:00-07:00), and the striatum were dissected and striatal DA and DOPAC were measured by HPLC with ECD. [Results & Conclusion] The activities of wheel running and striatal DOPAC/DA ratio significantly decreased in TSOD mice compared to TSNO control mice. These results suggest that the decreased spontaneous motor activity is also one of cause for obesity in addition to hyperphagia.

1P053-S Effect of pair-feeding on obesity in monosodium glutamate-induced obese mice

○Nobuhiro Yamamoto,Atushi Tohei,Yosuke Yanagawa,Hiromi Amao

Laboratory of Experimental Animal, Nippon Veterinary and Life Science University, Tokyo, Japan

Introduction An experimental obese animal model has been reported by injecting monosodium glutamate (MSG) to a mouse. We produced MSG mice using ddY mice and confirmed that the ddY-MSG mice exhibited obesity with hyperphagia, hyperinsulinemia and hyperleptinemia. In this study, to clarify whether hyperphagia is the sole cause of obesity in ddY-MSG mice, we examined the effect of pair-feeding on obesity in ddY-MSG mice. Materials and Methods MSG or saline was subcutaneously treated with newborn (within 24h of birth) male ddY mice. Saline-treated mice (Control group), MSG-treated mice fed ad libitum (AL group) and MSG mice pair-fed to control group between 3 to 10 weeks of age (PF group) were measured body weights, fat weights and blood glucose. Results Body weights, fat weight and blood glucose in AL group showed significant increases compared to those in Control and PF group. Body weights and blood glucose in PF group did not showed differences those of Control group. However, fat weight in PF group showed significant increases compared to that in Control group. Discussion Suppression of hyperphagia in ddY-MSG mice was effective at overweight and improving hyperglycemia, but did not affect fat weights. These results suggest that hyperphagia is not the sole cause of obesity in ddY-MSG mice. Factors including disorder of metabolism or spontaneous behavior may also be responsible for obesity in ddY-MSG mice.

S 113 1P054-S Macrophage USP2 potentially modulates type2 diabetes 1 2 1 3 ○Hiroshi Kitamura ,Shunsuke Kimura ,Tomomi Miyamoto ,Jun Okabe ,Yoshinori Shimamoto4,Yoshinori Naoe5,Ichiro Miyoshi1 1Graduate School of Medical Sciences, Nagoya City University, Nagoya Japan 2Graduate School of Medicine, Hokkaido University 3Backer IDI Heart and Diabetes Institution 4Kitasato University School of Veterinary Medicine 5National Center for Geriatrics and Gerontology

We have recently demonstrated that ubiquitin specific peptidase (USP) 2 suppressed expression of type 2 diabetes (T2D)-associated genes such as aP2, PAI-1, and MCP-1. In this study, we further analyzed pathophysiological roles of USP2 in T2D using gene-engineered cells and mice. Expression of the T2D-associated genes in USP2 knockdown (KD) macrophage-like cells were decreased by introduction of the longer variant of USP2 (USP2A) not but by the shorter variant (USP2B). Supernatant of USP2 KD cells induced inflammatory genes in 3T3-L1 adipocytes, followed by decrement of insulin sensitivity. High fat diet-induced obese Usp2a transgenic mice represented decreased accumulation of macrophages in mesenteric adipose tissue, in coincident with decreased expression of aP2 and PAI-1 in adipose tissue macrophages. Forced expression of Usp2a in macrophages ameliorated obese-induced insulin resistance. Moreover, expression of USP2A was significantly decreased in macrophages isolated from adipose tissues of ob/ob mice, while aP2 and PAI-1 was apparently increased. These results collectively indicate that macrophage USP2A potentially represses progression of T2D through modification of meta-inflammatory properties of macrophages.

1P055-S Osteoclast differentiation and function in CD98hc-deficient macrophage 1 1 2 3 ○Hideki Tsumura ,Naoko Ohnami ,Masamichi Takami ,Xiaokang Li ,Morihiro Ito4,Yasuhiko Ito4 1Division of Laboratory Animal Resource, National Research Institute for Child Health and Development, Tokyo, Japan 2School of Dentistry, Showa University, Shinagawa, Tokyo, Japan 3Division for radiation Safety and Transplantation Immunology, National Research Institute for Child Health and Development, Tokyo, Japan 4Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Aichi, Japan

CD98hc is highly expressed on proliferating lymphocytes and on other rapidly growing cell, indicating that CD98 is one of the important molecules for development, cell differentiation, cell proliferation, and cell fusion. We showed previously that the conventional gene-targeting of CD98hc lead to an early embryonic lethality. To investigate the role of CD98 in osteoclast, we have used Cre/loxP technology to enable the inactivation of tissue-specific CD98. CD98hc expression of CD98hclox/lox Lys-M cre mice decreased in peritoneal macrophages. To confirm the involvement of CD98hc in osteoclast formation, the peritoneal macrophages were stimulated with RANKL and mCSF, or co-culture with osteoblasts in the presence of 1,25(OH)2 vitamin D3 and then these cells were stained with tartrate-resistant acid phosphatase. The multinucleated osteoclast formation was severely impaired in peritoneal macrophages isolated from CD98hclox/loxLys-Mcre mice compared with those from CD98hcflox/flox mice. These results suggest that CD98 plays an important role in the osteoclast formation.

S 114 1P056-S Gamma-Aminobutyric Acid (GABA)-enriched brown rice depresses the elevation of blood pressure in SHR 1 2 1 3 4 ○Kohei Kawakami ,Kazuo Yamada ,Takaya Yamada ,Masato Nomura ,Toru Nabika 1Department of Experimental Animals, Shimane Univ., Izumo,Japan 2Department of Biochemistry, Shimane Univ., Izumo 3Department of Biotechnology and Chemistry, School of Engineering, Kinki Univ., Hiroshima 4Department of Functional Pathology, Shimane Univ., Izumo

γ-aminobutyric acid (GABA), ubiquitous non-protein amino acids known to be an inhibitory neurotransmitter, has attracted much interest due to its antihypertensive effect. If dietary intake of GABA can be increased through dietary means, it may provide substantial benefit to public health by lowering the incidence of hypertension. In the present context, we investigated the effect of GABA-enriched brown rice on blood pressure in SHR. As the average intake of rice by a standard adult Japanese was estimated 450 g/day, rats were fed 1.1 (low-GABA, comparable with 450 g/day in humans) and 2.1 g/day (high-GABA) of the GABA-enriched rice through a gastric tube (resulting in a GABA intake of 0.52 and 0.99 mg/day, respectively). Corn starch was given to control rats. Systolic blood pressures (SBP) was checked once a week. SBP was significantly lower in the rats fed with the GABA-enriched rice than in the control rats after 3 weeks of treatment (167.0±0.9, 168.5±2.2 and 181.4±1.6 mmHg for the high-GABA, the low-GABA and the control, respectively, P<0.01). BW and food consumption were not different among the three groups. These data indicated that a diet of GABA-enriched rice at a normal level of consumption could ameliorate high blood pressure.

1P057-S Reduction of the stress response by hiba essential oil inhalation in rats 1,2 1 2 ○Tetsuya Matsuura ,Takuya Tamaguchi ,Youhei Zaike ,Kousei Yanagihara2,Mitsuyuki Ichinose1,2 1Dept Chem Bioeng, Grad Sch Eng, Iwate Univ, Morioka, Japan 2Dept Welfare Eng, Iwate Univ, Morioka, Japan

To verify the effects of hiba essential oil in stressed rats, we analyzed physiological variables and psychophysiological behavior at 5-14 weeks. Rats were divided into three groups: nonstress control group (control rats), restrained stress group (stressed rats), and stress group subjected to hiba essential oil inhalation (stress-HEO rats). The paws of stressed rats were lightly restrained with rubber bands (three times/week). Subsequently, stress-HEO rats inhaled oil aroma for 30 min in a capped container. The quantities of food and water intake and the excretion amount of stressed rats were smaller than those of control rats during the experimental period. Body weights of stressed rats decreased compared with those of control rats. These physiological variables of stress-HEO rats significantly recovered compared with those of stressed rats (p<0.001). Adrenal gland enlargement and thymus atrophy were confirmed in stressed rats, although recovery of changes in anatomical variables was not observed in stress-HEO rats. Stress-related anxiety was assessed using the elevated plus-maze test. Entry times into the open arms of stressed rats were less than those of control rats (p<0.05). The suppression of entry times into the open arms of stressed rats was restored by hiba oil inhalation. The results suggest that hiba essential oil inhalation reduced stressed-induced growth inhibition and stress-related anxiety.

S 115 1P058-S Assessment of autonomic nervous function by tone-entropy analysis in NIBS miniature pigs 1 1 1 2 ○Masayoshi Kuwahara ,Masayuki Tanaka ,Koichi Ito ,Wataru Horii ,Koichi Katagiri2,Toshiki Saitoh2 1Department of Veterinary Pathophysiology and Animal Health, Graduate School of Agricultural and Life Sciences, The University of Tokyo 2Nippon Institute for Biological Science, Yamanashi, Japan

The aim of this study is to assess the autonomic nervous function in NIBS miniature pigs using tone-entropy analysis. Because tone-entropy analysis did not applied to animals until now, we also assessed and compared the autonomic nervous function in these animals by the conventional power spectral analysis of heart rate (HR) variability. For this purpose, ECGs were recorded from 6 NIBS miniature pigs using a Holter ECG recorder. ECGs were analyzed by ECG processor. ECG RR intervals are transformed into percentage index (PI) time series. The tone represents the balance between accelerations and inhibitions of the heart. The entropy was defined on PI distribution. Spectral powers were evaluated low frequency (LF: 0.01-0.07 Hz) and high frequency (HF: 0.07-1.0 Hz) powers of range. HR had diurnal rhythm clearly. Interactive autonomic modulations were expressed by a curved path in tone-entropy space. It was found that the tone was high and the entropy was low in dark period compared with in light period. Results in power spectral analysis were supported these findings. In this study, we confirmed the usefulness of tone-entropy analysis to assess the autonomic nervous function in miniature pigs. Further, NIBS miniature pigs clearly show the diurnal rhythms of the autonomic nervous function.

1P059-S in vivo imaging of human tumor xenografts using near-infrared fluorochrome-conjugated probes 1 1 1 1 ○Hiroshi Suemizu ,Kenji Kawai ,Miyuki Kuronuma ,Yuichiro Higuchi ,Haruo Hashimoto1,Tomoyuki Ogura1,Toshio Itoh1,Erika Sasaki1,Masato Nakamura2 1Central Institute for Experimental Animals, Kawasaki, Japan 2Tokai University School of Medicine, Isehara, Japan

Aim: We developed a versatile method for the detection of xenograft tumors without the need for fluoroprotein expression. Materials and methods: Human pancreatic cancer BxPC-3 and colorectal cancer HCT 116 cells were injected into the subcutaneous spaces, liver or tail vein of 7-12 week-old BALB/cA Rag2null Il2rgnull nude (BRG nude) mice to generate tumor xenografts. An anti-HLA monoclonal antibody was labeled with near-infrared fluorochrome (NIR) XenoLight CF770 and used as NIR-probes to study whole-body imaging. Results and Discussion: NIR fluorescent signals were observed at the xenografts within 24 h of probe injection. The accuracy of tumor targeting was confirmed by the localization of the NIR-HLA probe within the tdTomato-HCT 116 xenograft. Furthermore, we succeeded to visualize the patient-derived xenograft (PDX) LC11-JCK (non-small cell lung cancer) using the NIR-HLA probe without any genetic modification. The antibody accumulation within the tumor is based on the enhanced permeability and retention (EPR) effect, because there was no significant difference in the NIR signal intensity of the region of interest between the NIR-HLA probe and the NIR-isotype control. These results suggested that in vivo imaging with NIR-macromolecule probe is a valuable tool for the detection of human tumors in experimental metastasis models.

S 116 1P060-S C57BL/6J mice are more susceptible to oncogenic K-rasG12V-mediated lung tumors than A/J mice

○Hiromitsu Saito,Noboru Suzuki

Department of Animal Genomics Institute Mie University Life Science Research Center

The high incidence of oncogenic K-ras mutations is observed in lung adenocarcinoma of both human cases and carcinogen-induced animal models. The process of an oncogenic K-RAS mediated lung adenocarcinogenesis can be dissected into two parts; pre- and post- K-RAS mutation. Adoption of transgenic lines containing a flox-oncogenic K-ras transgene remove the use of chemical carcinogens and enables us to directly study crucial events specifically in post- K-ras mutation without considering the cellular events involved with oncogenic K-ras mutation, e.g., distribution and metabolism of chemical carcinogens, DNA repair and somatic recombination by host factors. We have generated two strains C57BL/6J-Ryr2tm1Nobs and A/J-Ryr2tm1Nobs in which oncogenic K-RasG12V can be induced at virtually at any tissues. Upon K-rasG12V induction in lung epithelial cells by adenovirus-expressing Cre recombinase (Ad-Cre), the number of tumors in C57BL/6J-Ryr2tm1Nobs/+ mouse line was 12.5 times as many as that in A/J-Ryr2tm1Nobs/+ mouse line. Quantitative trait locus (QTL) analysis using a polymorphic marker of second intron of K-ras gene demonstrated that wild-type K-ras gene was not involved in the differential susceptibility between two lines. Thus, our two K-rasG12V mediated lung tumor models might constitute a genetic system that enables us exploring new modifier genes in post-K-ras mutation.

1P061-S Study of mouse cancer model under the enriched housing environments 1,2,3 2 2 4 ○Satoru Arata ,Mamiko Mochizuki ,Hideto Matsuhashi ,Jun Watanabe ,Seiji Shioda2,4,Toshimitu Yamaoka3,Yasutuna Sasaki3 1Center for biotechnology, Showa University, Tokyo, Japan 2Center for Laboratory Animal Science, Showa University 3Institute of Molecular Oncology, Showa University 4Department of Anatomy, Showa University school of Medicine

(Background) Boost to phylactic power against cancer incidence is important. Enriched housing environments (EE) suppress tumor growth as well as to boost physical and mental health in mice (Cao et al., 2010). EE stimulate hypothalamic BDNF production and resulted down regulation of leptin production in adipocytes seem to implicate in antitumor effects, however, details are still unclear. Here, to define the EE effects in cancer, we tried to establish the mice cancer model under the minimum EE. (Methods) C57BL/6 mice were breed and live in EE using only Mouse Igloo & Fast-trac (Animec) in normal space cage (1 pair breeding or 5 mice per cage). Control mice were breed and held in normal space cage without EE goods. Tumor models were prepared to inoculate 5x105 live LLC tumor cells subcutaneously, or intravenously.(Results and conclusion) Tumor size and the rate of tumor growth in EE group mice after LLC inoculation were suppressed as compared with in control group. And Brain BDNF expression up regulated in EE mice. Thus, our minimum EE cage was confirmed to be functional. And we are examining the detail mechanism of tumor resistance in the EE mice.

S 117 1P062-S Estimating serial transplantation timing by weight loss in humanized leukemia mice

○Kazuo Ushida,Yuko Yamamoto,Yuu Dobashi,Kiyoaki Katahira,Satoshi Waguri

Translational Research Center, Fukushima Medical University, Fukushima, Japan

The purpose of this study is to establish the simple method for estimating the appropriate timing of serial transplantation of leukemia in humanized mice based on body weight changes. Lymphocytes isolated from clinical samples of pediatric leukemia (B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, malignant T-cell lymphoma, acute myeloid leukemia(AML)) were transplanted to NOG(NOD/Shi-scid, IL-2Rγnull) mice via tail vein with 1×106 or 1×107 cells. Existence of leukemia cells was evaluated by flow cytometer (BD FACSCantoTMII) and blood smears under Giemsa staining. At necropsy, spleen and bone marrow were also analyzed by flow cytometer. Body weights were measured twice a week with using a laboratory balance (SP2001, OHAUS). In NOG mice, successful transplantations of pediatric leukemia cells excluding AML were confirmed. The beginning of weight loss seemed to correspond to estimated time of cell proliferation. Since significant weight loss and worsened general condition of mice were thought to be associated with the high engraftment rate of leukemia cells, we judged it as the appropriate timing for serial transplantation. From the results of flow cytometer and blood smear, it seems to be possible to estimate the appropriate timing of serial transplantation by weight loss in pediatric leukemia-bearing NOG mice.

1P063-S The Role of carbohydrate-ligands of hematopoietic stem cells on homing and engraftment

○Noriyoshi Hashimoto,Keisuke Tabata,Soichiro Takagaki, Toshikazu Nishie, Masahide Asano Division of Transgenic Animal Science, Advanced Science Research Center, Kanazawa University, Kanazawa, Japan

The ligands containing carbohydrate chains are known as important cell adhesion molecules between leukocytes and their target tissues. We previously reported that β1,4-galactosyltransferase (β4GalT) -I-deficient mice were impaired in inflammatory responses due to the reduced expression of selectin ligands. Furthermore, bone marrow transplantation (BMT) efficiency was extremely low when hematopoietic stem cells (HSCs) from β4GalT-I-deficient mice were transplanted intravenously. In this study using β4GalT-I-deficient mice, HSCs were injected directly into the bone marrow cavity (IBM-BMT), because intravenous BMT might be failed by eliminating HSCs with reduced surface carbohydrates during the circulation. However, IBM-BMT did not improve BMT efficency, indicating that β4-galactosylation of HSCs is essential for homing to the bone marrow niches. Sialic acid is a kind of acidic carbohydrates and well known to modify non-reducing terminal carbohydrates on glycoproteins and glycolipids. UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) is also known as the key enzyme of sialic acid biosynthesis. When intravenous BMT was conducted using GNE mutant-bone marrow cells, BMT efficiency was moderately reduced. This result indicates that the reduced BMT efficiency using β4GalT-I-deficient HSCs, at least in part, is due to the reduced sialic acid-containing carbohydrate.

S 118 1P064-S IFN-γ is a key factor for increasing the bone volume in DCIR-deficient mice 1,2 1 4 1 ○Tomonori Kaifu ,Takumi Maruhashi ,Guangyu Ma ,Rikio Yabe ,Akimasa Seno3,Yoichiro Iwakura1,2 1Research Institution for Biomedical Science, Tokyo University of Science 2JST, Japan 3The University of Tokyo 4Heibei Medical University Fourth Hospital, China

DCIR is a C-type lectin receptor (CLR) with a unique signal motif for initiating inhibitory signaling. DCIR was structurally regarded as an inhibitory-type receptor, but the physiological role in a body system remained unclear. We have revealed that using Dcir-/-mice, DCIR functions as a negative regulator in the immune system, and we reported in the recent conference that DCIR also had an important role in regulating bone metabolism. Although DCIR deficiency dramatically elicited in vitro osteoclast (OC) formation, Dcir-/- mice had the increased bone volume, with higher number of OCs and osteoblasts (OB). To understand the precise role of DCIR in bone metabolism, we dissect the molecular mechanism that underlies the increase of bone volume in Dcir-/- mice. Notably, we found that Dcir-/- mice had a tendency to produce IFN-γ in lymph nodes. We showed IFN-γ, an osteogenic factor capable of facilitating mineralization of OBs. Moreover, Rag2-/- Dcir-/-cancelled the increase of bone volume, indicating that IFN-γ could be released from lymphocytes, especially from T cells. These findings demonstrate that Dcir-/- mice are prone to IFN-γ-dominated cytokine milieu, which accelerates mineralization of OBs, resulting in the increased bone volume. Collectively, Dcir-/-mouse is a novel animal model for dissecting the regulation of bone metabolism.

1P065-S Phenotypic analysis of mouse cardiovascular-respiratory system in Japan Mouse Clinic 1 1 1 1 ○Osamu Minowa ,Yoshihiko Sagara ,Tomoko Kagami ,Eiji Oka ,Hideaki Toki1,Tetsuo Noda1,2,Shigeharu Wakana1 1RIKEN Bioresource center, Tsukuba, Japan 2JFCR, Cancer institute

Mouse circulation-respiratory system phenotype analysis conducted in the Japan mouse clinic (JMC) includes blood pressure, electrocardiogram, echocardiogram, and pulmonary function test. It has been agreed in International Mouse Phenotyping Consortium (IMPC) under the standardized criteria these analysis are indispensable. In this report we examined the factors that vary measured value of the analysis and the stable measurement conditions. In order to perform many phenotypic analyses in the same individual in the JMC phenotyping pipeline, it's necessary to reduce the load on the mouse, and required the method applied to be non-invasive. Further, since these physiological test methods have originally been developed for the examination of human, once applied to the mice, it presents a several challenges to meet, nevertheless it has a direct value on phenotyping of a human disease models. Carrying out these analysis as a part of our comprehensive pipeline, would be an effective way not only for increasing the sensitivity of detection of the abnormal phenotype but for understanding better the whole picture of the phenotype, leading to enhancement of values of the mice as disease models.

S 119 1P066-S The multidimensional assessment system for developmental disorder model in mice

○Ikuko Yamada,Tomoko Kushida,Misho Kashimura,Tomohiro Suzuki, Hideki Kaneda, Kimio Kobayashi,Ikuo Miura,Tamio Furuse,Shigeharu Wakana RIKEN BioResource Center, Tsukuba, Japan

In Japan Mouse Clinic, we are conducting systematic and comprehensive mouse phenotyping analysis system. The diagnostic criteria of the human psychiatric disorders were updated from DSM-IV to DSM-5 in 2013. In the DSM-5, dimensional diagnosis was added to the categorical diagnosis. Therefore, our behavioral assessment system also changed into more detailed and multidimensional system. Recently the number of patients of developmental disorder increased and it became the social problem in human case. The existing animal model of developmental disorders are targeting single symptom. But in the clinical cases, many patients show various and continuous symptoms. Therefore, we try to establish multidimensional assessment system for the developmental disorder model in mice. In this study, we will report about baseline data and evaluation of this system. C57BL/6JJcl was purchased from CREA Japan, inc. at 4 weeks old. Behavioral tests were conducted in order of following, Ultrasonic vocalization (0~2w), Light/dark transition test (6w), Open-field test (7w), Crawley's social interaction test (8, 9w), Home-cage activity test (10-11w), Y-maze test (12w), Fear-conditioning test (13w), Pre-pulse inhibition test (14w). Using this new pipeline, we got a stable data which has less variation between subjects. We are planning to carry out the verification about the validity of this pipeline by using genetically modified mice M-174.

1P067-S Measuring system of Ultrasonic-Vocalizations in Neonatal mice in Japan Mouse Clinic

○Misho Kashimura,Tomoko Kushida,Tomohiro Suzuki,Hideki Kaneda, Kimio Kobayashi, Ikuo Miura,Ikuko Yamada,Tamio Furuse,Shigeharu Wakana Technology and Developmental Team for Mouse Phenotype Analysis, RIKEN BioResource Center, Tsukuba, Japan

A systematic and comprehensive analysis of mouse phenotype has been performed in Japan Mouse Clinic(JMC). Now, we are revising the behavior-specific phenotyping pipeline for evaluating mutants from the diversified viewpoints (Yamada et al. JALAS.2014). This pipeline focuses on not only locomotor activity, emotional and learning behavior but also social behavior in mutants. Crawley's 3 chamber is performed as social behavior test in this new-phenotyping pipeline. Also, we perform ultrasonic vocalization (USV) test for interactional communication in neonates and mothers. The USV is known as communication tool in mice, and it is used for courtship, sexual behavior, and communication between mothers and their pups. Especially, USV that is induced by maternal separation is shown within a week after birth. In addition, recent studies reported that some mouse models of autism indicated reduced level of USV. Therefore, USV test were performed for collecting control data in JMC. We used neonatal mice of the two strains which are C57BL/6J and DBA/2J. In this test, each pup was separated from the dam and the number of USV for 5 min was recorded from PND1 to 7 and 14. For further analysis, we attempted to record the USV with a mouse model of developmental disorder on PND1, 4, 7and14. Then we will report about the evaluation of USV-recording test in our new pipeline.

S 120 1P068-S Data mining to explore novel mouse models for human disease 1 2 2 ○Nobuhiko Tanaka ,HIDEAKI TOKI ,HIROMI MOTEGI ,TOMOHIRO SUZUKI3,HIDEKI KANEDA3,IKUO MIURA3,IKUKO YAMADA3,TAMIO FURUSE3,KIMIO KOBAYASHI3,MAKI INOUE2,OSAMU MINOWA2,TETSUO NODA2,SHIGEHARU WAKANA3,HIROSHI MASUYA1 1Tech. and Dev. Unit for Knowl. Base of Mouse Pheno., BRC, RIKEN 2Team for Advanced Dev. and Eval. of Human Disease Models, BRC, RIKEN 3Tech. and Dev. Team for Mouse Pheno. Anal. :JMC, BRC, RIKEN

The mouse (Mus musculus) has been well-known as a principal animal model for investigating and understanding the mechanisms underlying human disease. Throughout this decade, the phenotype data will be made available for all mouse protein-associated genes due to the effort of the International Mouse Phenotyping Consortium (IMPC; http://www.mousephenotype.org). Here, we aimed to develop a workflow to discover novel knowledge on biological phenomena and to explore novel disease models by analyzing comprehensive mouse phenotyping data from Japan Mouse Clinic. First, we standardized phenotypic data by groups and individuals, respectively. Using these standardized values, we explored the most appropriate workflow to achieve this purpose. Because visualizing complicated phenotypic results facilitate data interpretation, we have used methods for data mining such as cluster analysis. As a result, we proposed some new knowledge on biological phenomenon and some strains as candidate disease model. Future, based on this workflow, we plan to develop a supporting tool for searching novel disease models and publish the tool on the Internet.

1P069-S Analysis of background data on mouse model for Duchenne muscular dystrophy: C57BL/10Sc-mdx 1,2 3 3 4 ○Masahiko Yasuda ,Tomoyuki Ogura ,Yuyo Ka ,Takayuki Gotoh ,Chie Shimomura4,Megumi Nishiwaki4,Nobuhito Hayashimoto2,Riichi Takahashi3,Kenji Kawai1 1Pathological Analysis Center, Central Institute for Experimental Animals (CIEA), Kawasaki, Japan 2ICLAS Monitoring Center, Central Institute for Experimental Animals (CIEA), Kawasaki, Japan 3Animal Resources Center, Central Institute for Experimental Animals (CIEA), Kawasaki, Japan 4Technical Service Dept., CLEA Japan, Inc., Fujinomiya, Japan

CIEA has maintained the model mouse strains of muscular dystrophy, and has supplied them to research institutions. This time, in order to conribute to muscular dystrophy research, we analyzed background data on the Duchenne muscular dystrophy mouse model strain from C57BL/10Sc-mdx (mdx) mice against those from C57BL/6JJcl (B6) mice (20 animals/group/sex, 10 weeks of age). Background data included body weights, histopathology (femoris muscle, diaphragm and heart) and serum/plasma levels of CK, LDH, AST, ALT, creatinine, total protein, glucose, and potassium. Histopathology of skeletal muscle from mdx mice revealed dystrophic changes with active muscle fiber necrosis and regeneration. In addition, expression of dystrophin was not found in the skeletal muscle of mdx mice. Serum/plasma levels of CK, LDH, AST, ALT, and creatinine, from mdx mice, were much higher than those from B6 mice. Moreover, those levels from male mdx mice were higher than those from female mdx mice. Therefore, C57BL/10Sc-mdx mouse is an extremely useful animal model for Duchenne muscular dystrophy.

S 121 1P070-S Progranulin reduction affects tau phosphorylation in tau transgenic mice 1 1,2 1 1 ○Masato Hosokawa ,Tetsuaki Arai ,Masami Suzukake ,Hiromi Kondo ,Takashi Matsuwaki3,Masugi Nishihara3,Masato Hasegawa1,Haruhiko Akiyama1 1Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan 2University of Tsukuba, Tsukuba, Japan 3The University of Tokyo, Tokyo, Japan

Granulin (GRN) mutations were identified in familial frontotemporal lobar degeneration patients. GRN transcript haploinsufficiency is the proposed disease mechanism that leads to the loss of functional progranulin (PGRN) protein. Premature stop codons are not translated into the mutant transcript, which is lost by nonsense-mediated RNA decay. Recent findings have indicated that GRN mutations are associated with a wide spectrum of clinical phenotypes, including Alzheimer's disease and corticobasal degeneration. To investigate the influence of a decline in PGRN protein, P301L tau Tg mice harboring the GRN hemizygote were produced. Brains were collected from 13 or 19-month-old mice and then immunoblotting and immunohistochemical staining were performed. Immunoblotting analysis revealed that phosphorylated tau was increased in the Tris-saline soluble fraction of 13-month-old and in the sarkosyl-insoluble fraction of 19-month-old of P301L tau/GRN +/- as compared with that of the P301L tau Tg mice. Phosphorylated tau positive cells were relatively increased in 19-months old P301L tau/GRN +/- mouse of mid brain than that of P301L tau Tg mouse by immuno staining of phospho-tau antibody. The mechanism involved remains unknown, however, our data suggest that a reduction in PGRN protein might influence tau phosphorylation.

1P071-S Low MCV (mean corpuscular volume) value found in NER rat 1 1 1 1 ○Sho Takizawa ,Takahiro Yoshizawa ,Shin Shimada ,Norihisa Shiohara ,Tadao Serikawa2,Kiyoshi Matsumoto1 1Division of Laboratory Animal Research, Research center for human and environmental, Shinshu University, Nagano, Japan 2Kyoto University, Kyoto, Japan, and Osaka University of Pharmaceutical Sciences, Osaka, Japan

[Introduction] Noda epileptic rat (NER) is a strain that spontaneously exhibits generalized tonic-clonic seizures after 2-4 months of age. In this study, hematological analyses for NER were performed.[Methods] NER, Wistar and SD rats were used for hematological analysis. A complete blood count (CBC) was measured chronologically with XT-2000i (Sysmex) hemocytometer. Serological ferrokinetics data including serum ferritin, serum iron, total iron binding capacity (TIBC), and unsaturated iron binding capacity (UIBC) were also evaluated.[Results and Discussions] After 13 weeks of age, NER showed a low MCV value with decrease in red blood cell distribution width (RDW)-SD and increase in RDW-CV. Serum ferritin level was increased in NER, although mean corpuscular hemoglobin (MCH), serum iron, TIBC, and UIBC were not changed. These results suggest the possibility that low MCV value unaccompanied by anemia was a novel characteristic in NER. Additionally findings of transmission electron microscopy (TEM) observation and real-time RT-PCR in NER are introduced. It

S 122 1P072-S Gene expression profiling of sense and antisense transcripts in liver regeneration 1,2 3 2 ○Mitsuru Chiba ,Hiroshi Yasue ,Nobuhiro Ohkohchi

1Hirosaki University Graduate School of Health Sciences, Hirosaki, Japan 2Department of Surgery, University of Tsukuba, Tsukuba, Japan 3National Institute of Agrobiological Sciences, Tsukuba, Japan

Liver regeneration is a hyperplastic phenomenon induced by partial hepatectomy (PH) or hepatic damage. A large number of genes have been indicated to be involved in the process of liver regeneration. Recently, it has been reported that natural antisense transcripts are involved in the regulation of gene expressions. However, antisense transcript expressions in liver regeneration have not been reported to date. Therefore, the present study aimed to identify up-/down-regulated sense and antisense transcripts comprehensively in liver regeneration using PH mice and a sense/antisense custom-microarray. Ninety-seven genes were up-regulated and 7 genes were down-regulated for sense transcripts, whereas 15 genes were up-regulated and 2 genes were down-regulated for antisense transcripts in regenerating livers as compared to normal livers (p<0.05 and fold change>2.0). Of these genes, sense and antisense transcripts of 4 genes, Apoa4, Hp, Fgb, and Fgg, showed concordant up-regulation in the course of liver regeneration. Apoa4, Hp, and Fgb transcripts were further examined by strand-specific reverse transcription-quantitative polymerase chain reaction, revealing results consistent with those of the microarray. In conclusion, the up-/down-regulated sense and antisense transcripts identified in the present study were indicated to be involved in liver regeneration.

1P073-S Development and Mechanism Elucidation of Mouse Model for Priapism of Male Genital Disease 1 1 1 1 ○Hideki Kaneda ,Ikuo Miura ,Ryutaro Hukumura ,Tomohiro Suzuki ,Tamio Huruse1,Ikuko Yamada1,Kimio Kobayasi1,Hideaki Toki1,Yoichi Gondo1,Gen Yamada2,Shigeharu Wakana1 1RIKEN BioResource Center, Tsukuba, Japan 2Wakayama Medical University, Wakayama, Japan

In human, male genital diseases are general term for several diseases including prostate disease, erectile dysfunction and testicular tumor. In particular, male genital diseases are associated with symptoms of functional and/or morphological aberration in male genital system. Mouse models are expected to become the key to the treatment of intractable illnesses such as male genital diseases. In our project, we have been aimed to develop mouse mutants by genome-wide screening for various phenotypes observed in ENU-mutagenized mice to provide resources for studying the functions of genes, and to establish animal models for human diseases. In the recessive screening, we found out that the mutant family with symptoms identical to human male genital diseases, priapism. The phenotype state with onset usually around the 6 weeks, remain in maintenance of erection. Furthermore, this mutant showed body size, gait abnormality, defect in spermatogenesis and histopathological abnormality in penis. We begin more elaborate studies about histological analysis for male genitals and reproduction-related analysis. At the same moment, to gene identification, we have been next-generation sequencing. We report here on recent progresses concerning the above experimental results.

S 123 1P074-S Molecular Mechanism for Folliculogenesis, start from Transcription Factor Sohlh1 in mouse ovary 1 2 1 ○Hitomi Suzuki ,Aleksandar Rajkovic ,Masami Kanai

1Dept. EAMHD, TMDU, Tokyo, Japan 2Dept. OBGYN and RS, Univ of Pittsburgh, PA, USA

Folliculogenesis in mammals is a complex process involving bi-directional communication between the oocyte and surrounding soma. In the mouse ovary, primordial follicles (PrdFs) that contain 15um oocyte surrounded by flatten soma are formed shortly after birth, and provide a source of future growing follicles such as primary follicle (PrmF) during the entire reproductive lifespan. PrdFs remain dormant for prolonged intervals. For retaining a long female reproductivity, it is important to keep an appropriate balance of "dormant" and "activated" state of follicles. Sohlh1 and Sohlh2 are bHLH type transcription factors that are expressed specifically in oocyte of PrdF and early PrmF in mouse ovary. Knockout (KO) ovaries of those show the defects of premature ovarian insufficiency, loose most oocytes in a week after birth. We analyzed the KO mice and revealed that mutant follicles did not retain a dormant state and undergo an insufficient activated state resulting in massive oocyte death. Therefore, Sohlh1 and Sohlh2 are important for maintenance and survival of follicles. However, either Sohlh1 or Sohlh2 is expressed in both dormant and activated follicles, and the gene expression patterns, which discriminate these two types, are not reported yet. Thus we identified the new Sohlh1 binding factors that regulate Sohlh activities. In a meeting, we will show our results and are willing to discuss about possible functions.

1P075-S The effect of Sox17 on uterus during mouse implantation 1 1 2 1 ○Miyuri Kawasumi ,Hitomi Suzuki ,Yoshiakira Kanai ,Masami Kanai

1Tokyo Medical and Dental University, Tokyo, Japan 2Tokyo University, Tokyo, Japan

A critical event in the establishment of a successful pregnancy is in which the blastocyst attaches to the luminal epithelium of the uterus and invades into the stroma. It is well established in vitro fertilization and embryo transfer techniques on mice, however, treatments are unclear. Our aim is revealing the mechanism of embryo those implantation in vivo and in vitro and maintenance of pregnancy using Sox17 knockout mice and Sox17 mutant mice. Sox17 have reported their important role in endoderm development in the mouse. Sox17 was first detected in the nuclei of a subset of ICM cells, then detected only at the primitive endoderm cells of blastocyst. Also Sox17 strongly expressed at oviducts and uterus of adult mice. However, we do not know how it performs in those tissues. Although expression of E-cadherin was unaffected, integrinβ1 was reduced in Sox17 hetero gallbladder explants. Integrin is a major receptor for trophoblast-laminin interactions during ovulation and implantation and yolk sac placenta formation and it may be associated with Sox17 in the uterus at the time of implantation. Here, we study expression pattern and the role of Sox17 in embryo, oviduct, placenta and uterus during implantation in vivo environment.

S 124 1P076-S Experimental factors affecting onset time of anti- Thy1.1 antibody induced glomerulosclerosis in rats

○Mio Hiramatsu,Tasuku Nakatogawa,Mio Kawai,Akiko Uno, Natsuko Komiyama, Manabu Hirabayashi,Kumiko Koguchi,Tatsuo Yata,Hajime Sano,Mari Kinoshita, Eiji Kaneko, Masahiko Matsumoto

Chugai Research Institute for Medical Science, Inc., Shizuoka, Japan

Aims: The uninephrectomized rat with anti-Thy1.1 antibody administration (UNX-Thy1) is known as a model of chronic glomerulonephritis (CGN). In our conventional studies, Wistar rats are anesthetized with pentobarbital (PB) in uninephrectomy (UNX) followed by administration of anti-Thy1.1 antibody; however, onset time of CGN varied widely. To investigate experimental factors affecting the onset time, we focused on anesthesia, rat strain and timing of anti-Thy1.1 antibody administration. Methods: Male 5-7-week-old Slc:Wistar and F344/DuCrlCrlj rats were used. UNX was performed under PB (48 mg/kg, i.p.) or isoflurane (IF) anesthesia. CGN rats were established by an i.p. or i.v. injection of anti-Thy1.1 antibody (OX-7, 1 mg/kg) after UNX. We evaluated mainly changes in blood creatinine and hemoglobin. Results: In UNX-Thy1 model, there was no effect of anesthesia (PB vs IF) on onset time of CGN. The onset time didn't vary so widely in F344 rats compared to Wistar rats In addition, CGN rats administered anti-Thy1.1 antibody 2 weeks after UNX showed less variation in the onset time than immediately administered rats. Conclusions: In this study, it was considered that the experimental factors affecting onset time of CGN were rat strain and timing of anti-Thy1.1 antibody administration.

1P077-S Relation of atherosclerosis to the onset of coronary spasm and spastic angina in WHHLMI rabbits 1 1 1 1 ○Tomonari Koike ,Satoshi Yamada ,Shiori Tamura ,Ying Yu ,Nobue Kuniyoshi1,Masashi Shiomi1,2 1Institute for Experimental Animals, Kobe University Graduate School of Medicine, Kobe, Japan. 2Division of Comparative Pathophysiology, Kobe University Graduate School of Medicine

Purpose The mechanism of coronary spasm is still unknown. We examined relation of atherosclerosis to the development of coronary spasm using WHHLMI rabbits, an animal model for coronary atherosclerosis and myocardial infarction. Methods Coronary spasm was provoked in WHHLMI and normal Japanese white (JW) rabbits by bolus injection of ergonovine during norepinephrine infusion. Development of coronary spasm was evaluated by Electrocardiogram (ECG). Myocardial damage was evaluated by echocardiogram and serum markers for myocardial ischemia. Histopathological sections of coronary arteries were prepared. Results By pharmacological treatments, ECG showed ischemic changes in 91% of WHHLMI rabbits and 29% of JW rabbits. These changes were observed in every WHHLMI rabbits showing >75% cross-sectional narrowing (CSN) in coronary arteries but was 70% in rabbits showing <75% CSN. The maximum CSN was 81% in positive rabbits and 57% in negative rabbits. In these positive rabbits, the left ventricle mobility evaluated with echocardiogram was decreased. Every serum ischemic markers increased markedly 4 hours after vasospasm provocation. Conclusions These results suggest that the onset of coronary spasm was promoted on atherosclerotic coronary arteries and the WHHLMI rabbit is a good animal model for coronary spasm and spastic angina.

S 125 1P078-S Production of fibrillin 1 gene knockout cloned pigs by zinc finger nuclease 1,2 3 1,2 3 2 ○K Umeyama ,K Watanabe ,M Watanabe ,K Horiuchi ,K Nakano ,H Matsunari1,2,M Nagaya1,2,K Kosaki4,M Saito5,H Nagashima1,2,M Matsumoto3 1MUIIBR, Kawasaki, Japan 2School of Agriculture, Meiji University, Kawasaki, Japan 3Keio University School of Medicine, Tokyo, Japan 4Keio University School of Medicine, Tokyo, Japan 5Department of Restorative Dentistry, Tohoku University, Sendai, Japan

[Purpose] We sought to create a pig model for Marfan syndrome (MFS) by zinc finger nuclease (ZFN) to knockout the gene fibrillin 1 (FBN1), the causative gene for the syndrome. [Methods] ZFN mRNAs that targeted exon 10 of FBN1 were inserted into male porcine fetal fibroblast cells by electroporation and a cell line, designated F047 (+/Glu433AsnfsX98), was established as nuclear donor cells. [Results] In total, 206 blastocysts were obtained by somatic cell nuclear transfer and transferred into two recipient gilts, which became pregnant and gave birth to seven live and one stillborn pig. We then analyzed the phenotypes of the KO cloned pigs: two pigs (one live and one stillborn) had cleft palate; two live born pigs had pectus excavatum; computed tomography showed that one live born pig had delayed mineralization at the epiphyseal nuclei and primary trabeculae; disrupted elastic lamellae were present in the proximal thoracic aortas of some KO pigs. Thus, although the KO cloned pig siblings had an identical genetic background, they did not exhibit a consistent phenotype. [Summary] Mutation of FBN1, the causative gene for MFS in humans, produced phenotypic effects in KO cloned pigs that are similar to those of familial MFS in humans.

1P079-S Analysis of small testis mutant found in the common marmoset colony 1 1 1 1,2 ○Shuji Takabayashi ,Shiori Urano ,Narumi Otsubo ,Hideki Katoh

1Institute for Experimental Animals, Hamamatsu University School of Medicine, Hamamatsu, Japan 2Central Institute for Experimental Animals, Kawasaki, Japan

The common marmoset (hereafter marmoset) breed well in captivity with good reproductive efficiencies and their sexual maturity is reached within 18 months of age. It is necessary to grasp the testicular volume of the maturity marmoset to have relation to reproduction and the testicular size. Recently, we found mutant marmosets (SI018 and YI393) with small testes in our colony. In this study, we describe normal testicular volume and the phenotypes of mutant marmosets. The normal reference data were obtained from 38 mature males. The testicular volume were measured by ultrasonography and a plasma testosterone (TES) value were measured by automated immunoanalyser. The mean of the right and left testicular volume were 438.8±133.3 mm3 and 433.1±113.3 mm3, respectively. The mean of the TES value was 54.8±35.3 ng/mL. Whereas mutant marmosets had small testes and showed low value of plasma TES level. The left testis volume of SI018H and YI393 were 76.9 mm3 and 86.8 mm3, respectively. The TES level were 0.56 ng/mL and 1.45 ng/mL, respectively. Histological studies using mutant testis revealed a decrease in spermatogonia number and the differentiation abnormality of the spermatocyte. A normal sperm was not observed. It was found that SI018 and YI393 had the same ancestors as a result of pedigree analysis. It was suggested that these abnormality testis character had hereditary.

S 126 1P080-S Blood biochemistry and body fat distribution in spontaneous obese common marmosets 1 1,2 1,3 1,3 ○Takashi Inoue ,Keigo Hikishima ,Tomoko Ishibuchi ,Sayaka Ohba ,Norio Okahara1,Hideyuki Okano2,Toshio Itoh1 1Central Institute for Experimental Animals, Kawasaki, Japan 2School of Medicine, Keio University, Tokyo, Japan 3JAC Inc., Tokyo, Japan

Metabolic syndrome (MS) is considered as the concomitant clustering of obesity, dyslipidemia, hypertension, and glycaemia. In recent decades, the MS has become a leading health concern due to its link to cardiovascular disease. For pathoetiology and preclinical study of this syndrome, useful nonhuman primate models are necessary because of their similarity to human in metabolism and behavioral physiology. In this study, spontaneous obese common marmosets (Callithrix jacchus), small-bodied New World primates, were analyzed by blood chemistry test and MRI body fat measurement to investigate their ability as MS model animals. Four adult male and four adult female marmosets (3-8 years old) that have the body weight (447-493 g; mean 476 g) of 25% more than standard weight as spontaneous obese animals and another four males and four females of the same age that have the standard weight (326-363 g; mean 345 g) as normal control animals were used for analysis. Blood chemistry test showed that all obese marmosets had high triglyceride concentrations and most of them had high total cholesterol, γ-GTP, and GPT concentrations. Body fat measurement by MRI showed obese animals had significantly more abdominal visceral fat than normal. Hypertriglyceridemia and visceral fat accumulation of obese marmosets may suggest that this species become a useful model of the MS.

1P081-S The role of Regulator of G protein signaling 19 (RGS19) regulates the cell proliferation and differentiation of embryonic stem cell

○Seo Jin Park, Jae Woong Ryoo Kyungpook national university (South of Korea)

Mouse embryonic stem (ES) cells self-renew and are pluripotent, with the ability to differentiate into three layers and form all embryonic tissues. These ES cell abilities are maintained by intrinsic factors and an extrinsic factor. Many studies have contribute to the understanding of the molecular mechanisms involved in pluripotency maintenance, as well as controlled differentiation of ES cells to facilitate cellular therapy for disease. There is current interest in understanding cell cycle regulation in pluripotency and differentiation of ES cells. Progression through the cell cycle depends on the involvement of hetero-dimeric kinases composed of a regulatory subunit. The regulator of G protein signaling (RGS) family consists of more than 20 members. RGS19, an RGS member, specifically interacts with Gαi and Gαq to enhance their GTPase activity. Since growth factor receptors use Gi proteins to signal transduction, RGS19 might be involved in regulation of cell proliferation. In a previous gain-of-function study, RGS19 overexpression enhanced the cell proliferation of various cell types. Our data demonstrate that Rgs19 plays a role in the regulation of ES cell self-renewal and differentiation. Based on the existence of Rgs19 in ES cells, we assessed the role of Rgs19 in maintenance of self-renewal, and further compared morphological and molecular differences between wild-type and Rgs19 knock-out (KO) ES cells during LIF withdrawal and in vitro differentiation and teratoma formation. These findings provide insight for the first time into the mechanism involved in Rgs19 regulation of mES cell proliferation and differentiation.

S 127 1P082-S Signal responsiveness affected by genetic background in mouse embryonic stem cells

○Satoshi Ohtsuka,Hitoshi Niwa

Lab. for Pluripotent Stem Cell Studies, RIKEN CDB, Kobe, Japan

Robust self-renewal of embryonic stem cells (ESCs) is highly dependent on culture context and genetic background, which discriminates permissive and non-permissive genetic backgrounds for ESCs derivation. Recently, presence of two inhibitors (2i) in culture allows every rodents derived ESCs self-renewal. We revisited LIF responsiveness in ESCs, resulting that LIF responsiveness is highly variable dependent on the genetic background. In permissive strains derived ESCs (Sv, BL6 and BALB) showed that LIF strongly induced STAT3 targets with weak induction of MAP kinase target genes. However, ESCs from non-permissive strains (NOD, CBA and FVB) showed the opposite tendency, suggesting that permissiveness for ESCs derivation and maintenance might be dependent on a balance between STAT3 and MAP kinase pathway. Moreover, the balance altered by either increased STAT3 activity or inhibition of MAP kinase allows non-permissive strain derived ES cell lines self-renewing in conventional context. Collectively, our results establish that balance between STAT3 and MAP kinase branch is one of critical factors for permissiveness of ESCs self-renewal. Taken together, genetic background in ESCs influences growth factor responsiveness but also permissiveness for derivation of ESCs, which may be dependent on ratio between STAT3 vs MAP kinase activities.

1P083-S Maintenance of pluripotent stem cells controlled by Klf5 1,2 2 1 1,3 ○Takuya Azami ,Ken Matsumoto ,Hyojung Jeon ,Satoru Takahashi ,Masatsugu Ema2,4 1Dept. of Anatomy and Embryology, Faculty of Medicine, Univ. of Tsukuba 2Research Center for Animal Life Science, Shiga University of Medical Science 3Laboratory Animal Resource Center, University of Tsukuba 4PRESTO, JST

Pluripotency is maintained by the core transcription factors, such as Oct4, Sox2, and Nanog. These factors also have important roles in embryonic development and can reprogram somatic cells into iPS cells. Among the Yamanaka 4 factors, Klf4 is not indispensable for ES cells self-renewal and early embryo development. Klf4 together with Klf2 and Klf5 play redundant functions in ES cells. However, our previous study clearly indicated that Klf5 is indispensible for blastocyst development (Ema et al., 2008). Therefore, it is important to elucidate the molecular mechanism underlying these functions. To understand the mechanism, we amplified the cDNA from Klf5 KO and WT embryos and performed the microarray analysis. Then we found that intracellular signaling pathway A was activated in Klf5 KO embryos. Pharmacological inhibition for this pathway by specific inhibitor resulted in significant rescue for Klf5 KO phenotypes. Interestingly, Nanog positive pluripotent cells were emerged in ICM of Klf5 KO embryos. We conclude that Klf5 suppress this pathway and act as a safeguard for the normal cell-cycle progression and differentiation.

S 128 1P084-S Application of BAC-based gene targeting methods to rat ES cells 1 1 1 1 ○Iijima Saori ,Seiya Mizuno ,Yoko Tanimoto ,Yoko Daitoku ,Satoko Nakamura2,Mitsuko Sutoh2,Ryouji Hokao2,Fumihiro Sugiyama1,Ken-ichi Yagami1 1Laboratory Animal Resource Center, University of Tsukuba, Tsukuba, Japan 2Institute for Animal Reproduction, Kasumigaura, Japan

Studies in gene-targeted rats are expected to provide new insights in biomedical research. Therefore, we have been engaged in research to improve the technical basis for generation of gene-targeted rat strains. Here, we applied BAC targeting methods to rat ES cells. Previously, we reported a BAC-based gene targeting vector (rNV-BAC vector), which was designed to insert the fluorescent Venus reporter cassette into the endogenous Nanog locus. First, in the present study, we performed transgenic experiments to examine the accuracy of this rNV-BAC vector. The Venus-reporter system to monitor Nanog gene expression was functional not only in mouse ES cells but also in rats in vivo. Next, to determine whether BAC targeting is effective in rat ES cells, rNV-BAC vector was introduced into rat ES cells by electroporation. FISH analysis verified that the Venus-reporter cassette replaced the endogenous Nanog locus by homologous recombination. However, we could not obtain gene-targeted rat ES cells by conventional targeting methods using a plasmid-based targeting vector designed to introduce the same mutation into the Nanog locus as the rNV-BAC vector. The success of BAC targeting in rat ES cells will facilitate the generation of sophisticated gene-targeted rat strains in which to study human diseases.

1P085-T Production of the ApoE deficient hyperlipemic mice using CRISPR/Cas9 system 1 1 1 1 ○Takao Tanaka , Yoshihiro Ohguchi , Toshio Shimada , Kiyokazu Nakazato , Yoshiki Miura1, Motoharu Ido1, Kim JiRyun2, Aya Nakai2 1Bioscience Business Division, KAC Co., Ltd., Shiga, Japan 2Bio Division, APRO Science Inc., Tokushima, Japan

To generate homozygously ApoE (Apolipoprotein E) deficient hyperlipidemic mice in a short period, we employed CRISPR/Cas9 system. Though the sequence analysis of mutation sites has not been completed, we obtained hyperlipidemic mice without going through the process of mating male and female heterozygous mutants.

S 129 1P086-S Production of mutant mouse using by CRISPR/Cas system 1,2 2 3 4 ○Kazuki Nakao ,Hiroshi Kiyonari ,Yasuhide Furuta ,Rika Numano ,Takeshi Harada1,Harumi Nakao1,Atsu Aiba1 1The Laboratory of Animal Resources Center for Disease Biology and Integrative Medicine Faculty of Medicine, University of Tokyo, Tokyo, Japan 2Laboratory for Animal Resources and Genetic Engineering Animal Resource Unit, RIKEN, Kobe, Japan 3Laboratory for Animal Resources and Genetic Engineering Animal Resource Unit, RIKEN, Kobe, Japan 4the Electronics-Inspired Interdisciplinary Research Inst., Toyohashi Univ. of Technology

Generally, embryonic stem cells (ES cells) are necessary for generating of knock out (KO) mice. But it is costly and laborious to use ES cells. It was reported that KO mice were directly obtained from fertilized egg by using ZFN and TALEN. Recently, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system was reported as a new technology of genome editing. Bacteria and archaea have evolved an RNA-based adaptive immune system that uses CRISPR/Cas proteins to detect and destroy invading viruses and plasmids. Here, we show an efficient method to generate mutant mice by microinjection of Cas nuclease and single-guide (sg) RNAs into inbred mouse strain. Injection of Cas9 mRNA and sgRNAs into cytoplasm of pronuclear stage embryos generated approximately 60% of Grm1 mutant mice. From this result, we think the CRISPR/Cas system as one of the powerful tools of the mutant animal generation.

1P087-S Generation of knockin rats by CRISPR/Cas9

○Kazuto Yoshimi,Takehito Kaneko,Tomoji Mashimo

Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto, Japan

Recently RNA-guided nucleases CRISPR/Cas9 system was established as a newly efficient genome editing tool. Here, we employed CRISPR/Cas for accurate and efficient genome editing in rats. The synthetic chimeric guide RNAs (gRNAs) discriminated a single nucleotide polymorphism (SNP) difference in rat embryonic fibroblasts, allowing allele-specific genome editing of the dominant phenotype in (F344 x DA)F1 hybrid embryos. Using single-stranded oligodeoxynucleotides, we recovered three recessive phenotypes: the albino phenotype by a SNP exchange; the non-agouti phenotype by integration of a 19-bp DNA fragment; and the hooded phenotype by eliminating a 7,098-bp insertional DNA fragment, evolutionary-derived from an endogenous retrovirus. Successful in vivo application of the CRISPR/Cas system confirms its importance as a genetic engineering tool for creating rat models of human diseases.

S 130 1P088-S Applied Genome Editing with CRISPR/Cas9 Plasmid in Mice 1 1 1 1 ○Seiya Mizuno ,Kanako Kato ,Saori Iijima ,Huong Tra Dinh Thi ,Yoko Tanimoto1,Yoko Daitoku1,Miyuki Ishida1,Masatsugu Ema2,Masahito Ikawa3,Satoru Takahashi1,Fumihiro Sugiyama1,Ken-ichi Yagami1 1Laboratory Animal Resource Center, University of Tsukuba 2Research Center for Animal Life Science, Shiga University of Medical Science 3Research Institute for Microbial Diseases, Osaka University

The CRISPR/Cas9 system has been developed as a genome editing method. The CRISPR/Cas9 plasmid DNA vectors are very easily constructed. In addition, gene modified mice can be simply produced by microinjecting CRISPR/Cas9 plasmid DNA into pronuclear. For this reason, it is expected that CRISPR/Cas9 might be used for various scientific research. The knockout alleles are generated through site-specific double strand break repair, which causes unpredictable insertions or deletions of various sizes by non-homologous end joining. To generate optional single nucleotide mutation (SNM), we co-microinjected both the CRISPR/Cas9 plasmid and the single stranded DNA donor into mouse one cell embryos. As a result, we obtained the mice bearing designed SNM alleles by homology-directed repair. We also generated the reporter knock-in allele by co-microinjection of the CRISPR/Cas9 plasmid and the double stranded DNA donor. Moreover, mutant mouse with genomic deletion greater than 1 Mb in size was generated by co-microinjection of two different CRISPR/Cas9 plasmid vectors. These results suggest that combinations of the CRISPR plasmid vector and DNA donor, and two different CRISPR plasmid vectors are able to make more flexible genome editing in mice.

1P089-S Mouse mutagenesis by pronuclear injection of circular CRISPR/Cas9 plasmid into zygotes 1 1 2 2 1,2 ○Yoshitaka Fujihara ,Daisuke Mahiko ,Saki Nishioka ,Yoko Esaki ,Masahito Ikawa

1Research Institute for Microbial Diseases, Osaka University, Osaka, Japan 2NPO for Biotechnology Research and Development, Osaka, Japan

CRISPR/Cas mediated genome editing has been successfully demonstrated in mammalian cells and further applications for generating mutant mice were reported by injecting humanized Cas9 (hCas9) mRNA and single guide RNAs (sgRNAs) into fertilized eggs. When we randomly chose 291 target sequences within protein coding regions of 73 genes, an average number of off-target candidates (exact match 13 nucleotides from 3' target and NGG) found by Bowtie software was 9.2 ± 21.0 (~1.8 times more than the estimated value, 5.2). We next validated their activity by observing green fluorescence reconstituted by homology dependent repair (HDR) of an EGFP expression cassette in HEK293T cells. Of the pX330 plasmids expressing hCas9 and sgRNA tested, 81.8% (238/291) were found to be functional in vitro. We finally injected the validated pX330 plasmids into mouse zygotes in its circular form against 32 genes and obtained mutant mice at a 52.9 ± 22.3% (100/196) mutation frequency. Among the pups carrying mutations on the autosomes, 43.6% (47/96) carried the mutations in both alleles. When off-target candidate sites were examined in 63 mutant mice, 0.8% (3/382) were mutated. We conclude that our method provides a simple, efficient, and cost-effective way for mammalian gene editing that is applicable for large scale mutagenesis in mammals.

S 131 1P090-S Innate immune factor Lipocalin2 as an sperm maturation factor 1 2 3 2 ○Watanabe Hitomi ,Toru Takeo ,Hiromasa Tojo ,Kazuhito Sakoh ,Naomi Nakagata2,Mak Tak W.4,Gen Kondoh1 1Institute for Frontier Medical Sciences,Kyoto University,Kyoto,Japan 2Center for Animal Resources and Development, Kumamoto University, Kumamoto , Japan 3Graduate School of Medicine, Osaka University, Osaka , Japan 4University Health Network, Toronto, ON, Canada

Innate immune factor Lipocalin2 as an sperm maturation factorMammalian sperm undergo multiple maturation steps after leaving the testis in order to come competent for fertilization, although the molecular mechanisms underlying this process remain unclear. We found that lipocalin2 (Lcn2) can facilitate sperm maturation. Most sperm that migrated to the oviduct of wild-type female underwent lipid raft reorganization, but few did so in Lcn2 null mice. Furthermore, we found that LCN2 binds to membrane phosphatidylethanolamine (PE) and immediately induces lipid raft movement via PKA-dependent pathway. Finally, Lcn2 facilitated cholesterol efflux to complete capacitation process. These observations imply that mammals possess another mode for sperm maturation, different from the albumin-mediated pathway.

3P001-S Preservation effect of mouse spermatozoa by vacuum-drying and storing at RT in 3-O-methyl-D-glucose 1 1,2 ○Eri Nakamura ,Norihiro Tada

1Research Institute for Diseases of Old Age, Juntendo University Graduate School of Medicine 2Atopy Research Center, Juntendo University Graduate School of Medicine

We report the fertilizing and developmental ability of spermatozoa, vacuum-dried with Tris-EGTA-100µM epicatechin+100µM ascorbic acid (Tea solution) containing 1mM, 10mM or 100mM 3-O-methyl-D-glucose (3-OMG) in microtube, and then stored for 1 week at RT. After rehydration, spermatozoa were injected into oocytes by ICSI and embryonic development was followed. The percentages of fertilizing ability and 2-cell development of oocytes injected with spermatozoa vacuum-dried and preserved with Tea solution containing 1mM, 10mM or 100mM 3-OMG were 93.1%, 95.5% and 26.3% for fertilization, and 88.9%, 85.7% and 100% for 2cell development, respectively. The fertilization rates of oocytes injected with spermatozoa preserved with 100mM 3-OMG were significantly lower than that of spermatozoa preserved with 1mM and 10mM 3-OMG. However, there were no significantly differences among 3-OMG concentration in 2cell-developmental rates. On the other hand, the developmental rates of blastocysts were recognized only 1mM 3-OMG (3.7%). The percentages of fertilizing ability, 2-cell and blastocyst development of oocytes injected with spermatozoa stored for 4.5 years at RT were 95.6%, 86.0%, and 7.0%, respectively. Our data suggested that 3-OMG had no effect for vacuum drying spermatozoa and spermatozoa vacuum-dried with Tea solution and stored for 4.5 year at RT had fertilizing ability and embryonic development.

S 132 3P002-S Effect of lipids efflux from plasma membrane on fertility of mouse sperm 1 1 1 1 ○Toru Takeo ,Kazuhito Sakoh ,Satohiro Nakao ,Yuta Ishizuka ,Hidetaka Yoshimoto1,Yuka Horikoshi1,Yumiko Hirose1,Shiori Takeuji1,Ayumi Mukunoki1,Naomi Nakagata1 1Division of Reproductive Engineering, Center for Animal Resources and Development, Kumamoto University, Kumamoto, Japan

Membrane cholesterol efflux is involved in sperm capacitation in mammal. Treatment of cholesterol acceptors of bovine serum albumin (BSA) or methyl-β-cyclodextrin (MBCD) to sperm is applied to efficiently induce sperm capacitation in in vitro fertilization (IVF). It is known that the activation of sperm fertility by BSA or MBCD is mainly dependent on the ability of cholesterol removal. But the detail mechanism of cholesterol acceptors is not fully understood. In this study, we focused on the effect of BSA and various cyclodextrins (MBCD, dimethyl-β-cyclodextrin [DMBCD], and dimethyl-α-cyclodextrin [DMACD]) on lipid efflux from the plasma membrane of mouse sperm. In addition, we examined the fertilizing ability of sperm after exposure to the additives. BSA, MBCD, and DMBCD induced cholesterol efflux whereas DMACD did not. On the other hand, all the additives induced phospholipids efflux. The fertility of sperm was induced by treatment of all the additives. Interestingly, DMACD could not facilitate cholesterol efflux but enhanced sperm fertility. These results suggest a novel mechanism that is independent of cholesterol efflux, by which sperm capacitation is induced. In summary, we demonstrated that various cyclodextrins have the ability to activate sperm fertility via different mechanisms.

3P003-S Effect of bpV(pic) on superovulation in five strains of mice

○Osamu Suzuki,Minako Koura,Kozue Uchio-Yamada,Junichiro Matsuda Laboratory of Animal Models for Human Diseases, National Institute of Biomedical Innovation, Ibaraki, Japan

[Aim] Strain/individual differences in superovulation efficiency with gonadotropins constitute a serious problem in mouse reproduction (Suzuki et al. Reprod Fertil Dev, 8(6):975-980, 1996). To solve this problem, we focused on the role of the Phosphatase and Tensin Homolog Deleted from Chromosome 10 (PTEN) in folliculogenesis. In this study, the effect of a PTEN inhibitor on superovulation was compared between five strains of mice. [Method] Females at 28 days of age were injected with 0 (control) or 30 µg of dipotassium bisperoxo (picolinato) oxovanadate (V) (bpV(pic), Enzo) dissolved in Ringer's solution and 5 iu of PMSG. About 48 h later, 5 iu of hCG was injected to the females. About 16 h later, the numbers of ovulated oocytes per female were examined. [Results and Discussion] The average numbers of oocytes collected were 12.6±3.7 vs. 21.2±2.0, 23.0±8.4 vs. 30.8±6.0, 14.4±3.0 vs. 19.6±9.4, 10.8±3.4 vs. 9.6±1.7, 19.6±4.8 vs. 15.0±2.6 (control vs. bpV(pic), mean±SEM, n=5) in A/J, C57BL/6N, ICR, DBA/2, and C3H/HeJ, respectively. No significant difference between control and bpV(pic) groups in each strain was found on analysis of variance, but more oocytes tended to be collected in the bpV(pic) groups in A/J, C57BL/6N and ICR. Thus, our new method using both PTEN inhibitors and gonadotropins is a promising method for improving superovulation in mice, although the strain difference in the effect of bpV(pic) is not negligible.

S 133 3P004-S Examination of maternal control of the in vitro- matured mouse oocytes derived from GV stage oocytes 1 1 2 3 ○Megumi Sakita ,Miku Kamei ,Takao Nakagawa ,Manami Nishimura ,Masataka Nakaya3,Shintarou Kobayashi3,Rika Azuma3,Tasuku Mitani4,Hiromi Kato4,Masao Kishi4,5,Yoshihiko Hosoi1,4,Masayuki Anzai4 1Development of Genetic Engineering, Kinki University 2Kiwa Laboratory Animals Co., Ltd. 3Division of Biological Science, Graduate School of Biology-Oriented Science and Technology, Kinki University 4Institute of Advanced Technology, Kinki University 5Experimental Farm, Kinki University [Objective]The present study aimed to improve the developmental ability of in vitro matured oocytes.We first obtained the fetuses fromin vitro produced embryos by the ART using the GV oocytes of C57BL/6 inbred mice. In this study, we examined the accumulation of mRNA in oocytes and embryos to determine a control change of the transcription expression during in vitro maturation and fertilization. [Methos]Immature oocytes were collected after the PMSG dosage to C57BL/6J mouse in 46 hr and performed culture in mTaM for 16 hr. In vitro maturation and fertilization of oocytes were performed according to Nishimura et al. In situ hybridization was done for the collected GV stage, next matured MII stage and early embryos. [Results]In the result of in situ hybridization using QuantiGeneViewRNA, all oocytes showed an expression of Gdf9. Furthermore, the brightness detection of in vitro mature oocytes is significantly higher than in PN oocytes. Gdf9 which needs the selective decomposition for the mRNA was probably because Gdf9 is not synthesized in ZGA, the accumulation of Gdf9 cause effects on the inhibition of the generation.

3P005-S Study in recipe of progesterone to delay parturition of recipient mice for Caesarean section 1 1 2 2 ○Ayako Wada ,Megumi Ibayashi ,Mami Hayashi ,Kagari Kameda ,Satoshi Tsukamoto2,Seiji Kito2 1Science Service Inc. 2National Institute of Radiological Sciences

For microbiological cleaning of mice, we employ Caesarean section (C-section) to embryo transferred recipients. Because recipe of progesterone (P4) injection to delay parturition is yet to be determined, we tried to optimize timing and amount of P4 for the recipients. Embryos of C57BL/6JJmsSlc were transferred to Jcl:ICR(MCH) females on the day of vaginal plug formation (Day 1) and C-sectioned at 14:00 on Day 20. Amount and timing of P4 were 1, 2 or 3 mg at 13:30 on Day 19 and at 9:00 on Day 20 for multiple injections and 2 or 3 mg at 13:30 on Day 17, 18 or 19 for single injection, with 6-9 recipients assigned in each group. Delayed parturition failed in 1/7 mice with 1 mg on Day 19 and 20, in 1/7 mice with 3 mg on Day 17 and in 2/7 mice with 2 mg on Day 18. P4 injection did not affect fetal development to term and recovery at C-section. However, average recovery time periods (from omphalotomy to the beginning of pulmonary respiration) differed from 223 to 458 sec (p<0.05); shortest in mice injected 3 mg on Day 17 and longest in mice injected 3 mg on Day 19 and 20 with the tendency of shorter in mice injected earlier day and smaller amount. These results indicate significant effects of timing and amount of P4 on recovery time period and preferred injection of 3 mg on Day 28 or 2 mg on day 19. The results of other timing and amount, as well as results using recipients transferred frozen-thawed embryos will be also discussed.

S 134 3P006-S Effect of amino acid derivative addition to in vitro matured medium for mouse immature oocytes 1 1 2 3 ○Miku Kamei ,Megumi Sakita ,Takao Nakagawa ,Masataka Nakaya ,Manami Nishimura3,Rika Azuma3,Shintaro Kobayashi3,Tasuku Mitani4,Hiromi Kato4,Masao Kishi4,5,Yoshihiko Hosoi1,4,Masayuki Anzai4 1Development of Genetic Engineering, Kinki University 2Kiwa Laboratory Animals Co., Ltd. 3Division of Biological Science, Graduate School of Biology-Oriented Science and Technology, Kinki University 4Institute of Advanced Technology, Kinki University 5Experimental Farm, Kinki University

[Objective]In the mitochondria and cytoplasm transport pathway, amino acid derivatives such as carnitine are closely related to the fatty acid β-oxidation. In this study, we determined the effects of addition of L-carnitine to the in vitro matured medium on the early embryonic development of the mouse immature oocytes. [Methods]Adult female ICR were used in this experiment. Mice were injected with PMSG. After 46 hours, extirpated ovaries were finely cut, and COCs were collected from ovaries. Thereafter, immature oocytes were transferred to the mTaM with each concentration (1 - 10mM) of L-carnitine. Next, the in vitro maturation and fertilization was performed according of Nishimura et al. [Results]There was no significant difference in the capacity for the in vitro maturation and fertilization of oocytes between the addition and non-addition of L-carnitine in the matured medium. However, the blastocyst development in the addition group of L-carnitine (2 - 5mM) was higher as compared in the non-addition groups. These results, in vitro maturation medium containing amino acid was improving the generation of the early embryo.

3P007-S Effect of in vitro culture of an early embryo on development of a mouse 1 1 1 1 ○Kanako Oda ,Toshiya Sato ,Yoshitaka Maeda ,Seiko Sakai ,Nobuyoshi Fujisawa1,Minesuke Yokoyama1,2,Toshikuni Sasaoka1 1Animal Resources Branch Center for Bioresource-baced Researches Brain Research Institute,University of Niigata,Niigata,Japan 2Central Institute for Experimental Animals,Kawasaki,Japan

Reproductive engineering technology is widely used for production of mice. However, it remains unknown whether characteristics of in vitro cultured mouse embryos are the same as those of in vivo cultured mouse embryos. To clarify effects of the in vitro culture on mouse development, we examined in vitro cultured embryos with respect to morphology, developmental rate, cell numbers and amount of gene expression at blastocyst stage and measured body weight of mice derived from in vitro culture embryos and in vivo cultured embryos. In vitro cultured embryos were subjected to be cultured in KSOMaa supplemented with human recombinant albumin (rHA) or MW with rHA. In vivo cultured embryos were transferred into the oviducts of pseudopregnant females and harvested. We observed that the in vitro cultured embryos developed faster and contained fewer cells of inner cell mass than the in vivo cultured embryos. We found that number of pups from the in vitro cultured embryos was decreased at birth and body weights of mice derived from the in vitro cultured embryos were increased. Amount of H19 gene expression was decreased in the in vitro cultured embryos. These results suggest the in vitro culture of embryos has an influence on several characteristics in mouse development.

S 135 3P008-T Establish a new method of artificial insemination in mice with BMY method 1 1,2 ○Tsunekata Ito , Kazuo Ohwada

1Laboratory Animal Center, Institute for Promotion of Medical Science Research, Yamagata University Faculty of Medicine, Yamagata, Japan 2National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan Frozen sperm in mice is used actively in the transfer of institutions between. It is common case of restoration of individuals in mice using frozen sperm were thawed frozen sperm, fertilized with eggs, is to transplantation the living body the two-cell stage embryos obtained by culture for 24 hours thereafter. However, it is considered artificial insemination to be directly injected into female mice frozen sperm is efficient, artificial insemination in mice has not been popular. Therefore as a preliminary experimentation to establish the artificial insemination with frozen sperm in mice, we were induced pseudopregnancy during the day female mice treated with BMY (Breeding Method in Yamagata Univ.). Then we examined offspring whether efficiently obtained by the artificial insemination of fresh spermatozoa immediately using the ITS (intrabursal transfer of sperm) method. Copulation rate of group 2 artificial insemination, pregnancy rate, average implantation rate and average survival litter size was a good performance equal to or better than the control group. We compared the production index these results, good results were obtained than in the control group in the ITS-B group especially. Because it showed a good performance in the artificial insemination of mice with fresh sperm, we are expected to applying artificial insemination using frozen sperm future.

3P009-T Live mouse recovery rate from cryopreserved sperm and embryos in Lab. Anim. Resource Bank at NIBIO ○Minako Koura, Akiko Kawai, Masafumi Nakano, Yoko Noguchi, Osamu Suzuki, Junichiro Matsuda National Institute of Biomedical Innovation. Laboratory of Animal Models for Human Diseases.

In our recent activities concerning "Safe deposit support services" by Laboratory Animal Resource Bank at NIBIO, we are dealing with increasing numbers of frozen embryos and sperms prepared in universities and institutes, sometimes even in foreign countries. We are going to summarize the results of about 40 cases from 2009 to 2013, and explain the recovery ratio and methods.

S 136 3P010-S Storage of 2-cell embryos at 10°C for one day before transfer into transgenic mice

○Masahiro Morita,Mami Kakefuda,Hiromi Tateishi,Kaoru Matsumoto, Kiyoharu Sato, Chisato Goto,Satomi Uchida,Yosuke Kawase CHUGAI RESEARCH INSTITUTE FOR MEDICAL SCIENCE, Shizuoka, Japan

In vitro fertilization (IVF) and embryo transfer methods are useful for producing large numbers of transgenic mice. When there are not enough pseudopregnant recipient mice, embryos need to be stored for one day. We recently reported that low-temperature preservation was a useful method of short-term storage for 2-cell embryos (Conference for Laboratory Animal Sciences and Technologies, KYUSHU 2012). In this study, we demonstrated the effects of one-day storage on 2-cell embryos at 10°C using 10 lines of transgenic mice. The 2-cell embryos, which were produced by IVF using transgenic C57BL/6 or hybrid mice, were transferred to mice allocated to two experimental groups. I: Control (no short-term storage) II: 2-cell embryos were stored for one day at 10.0 ± 0.3°C in a low-temperature incubator in 20 mM HEPES-buffered Whitten's medium. The 2-cell embryos were transferred into the oviducts of 0.5 dpc pseudopregnant recipient mice. In 7 lines out of 10, the developmental rates to full-term were 40-51% (experimental group I) and 39-57% (experimental group II). These results were not significantly different, and 10°C preservation using a low-temperature incubator was demonstrated to be a useful method of short-term storage for 2-cell embryos in the large-scale production of transgenic mice.

3P011-T Development to offspring from embryos and sperm preserved in National BioResource Project - Rat

○Takehito Kaneko, Hiroaki Taketsuru, Tomoji Mashimo, Takashi Kuramoto

Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto, Japan

In the National BioResource Project-Rat (NBRP-Rat), 715 rat strains have been deposited so far. For all deposited strains, 2-cell embryos or sperm have been cryopreserved. Therefore, it is important to examine the developmental abilities of the preserved embryos and the sperm for supplying resources from NBRP-Rat. We here report the development to offspring from the embryos (Inbred: 19 strains, mutant: 2 strains and congenic: 1 strain) and the sperm (2 mutant strains) that were preserved in NBRP-Rat from April 2010 to March 2013. Two-cell embryos were cryopreserved using vitrification solution (PEPeS) which contained propylene glycol, ethylene glycol, percoll and sucrose. Sucrose solution was used for warming of the embryos. After warming, 60-100% of the embryos showed morphologically normal. Then, 36-100% of the embryos were transferred into the oviducts of pseudopregnant females, and 10-100% embryos developed to offspring. On the other hand, the sperm of 2 mutant strains were cryopreserved using Tris-EDTA buffer solution. After warming, oocytes were fertilized with the sperm by intracytoplasmic sperm injection. After embryo transfer, 12% and 31% of the embryos developed to offspring. In conclusion, offspring were obtained from the preserved embryos and sperm in all strains that examined in this study.

S 137

3P012-T A Study for Collection and Vitrification of Rat Blastocysts

○Tomoo Eto Central Institute for Experimental Animals, Kawasaki, Japan

To produce chimeric rats, embryonic stem (ES) cells, recipient female, and host embryos are needed. However, great efforts are necessary to reintegrate the unity as synchronizing the time schedules of these three kinds. Therefore, we studied collection and vitrification of the host embryos. Male and female rats of the Brl Han:WIST@Jcl(GALAS) strain underwent natural crossbreeding and the blastocysts were collected (group A). The eight-cell embryos were also collected and cultured in vitro to the blastocyst (group B). Vitrification was performed according to the method of Eto et al. [2014]. In addition, embryos were transferred without vitrification of blastocysts of group B (group C). The mean number of embryos collected per female rat was nine for eight-cell embryos and seven for blastocytes (group A). However, when the eight-cell embryos were developed to the blastocysts, the mean number of embryos collected decreased to seven per female rat (group B). The survival rate of embryos after vitrification was 93% in group A, 64% in B, and 64% in C. In group B, 28% of the blastocytes atrophied. After warming, normal blastocysts were used for embryo transfer and were then examined for development of live fetuses. The development rate of live fetuses was 49% in A, 47% in B, and 51% in C. The experimental results indicated that group A is efficient for making vitrified host embryos. In future, we will inject ES cells into the blastocysts to make chimeric embryos.

3P013-S Development of new seed mice with C57BL/6N genetic background used for targeted transgenesis

1 1 2 3 ○Masato Ohtsuka ,Hiromi Miura ,Ayako Isotani ,Masahito Ikawa ,Masahiro Sato4,Minoru Kimura1 1Sch. of Med., Tokai Univ., Kanagawa, Japan 2Immunol. Front. Res. Center, Osaka Univ., Osaka, Japan 3Res. Inst. for Microbial Dis., Osaka Univ., Osaka, Japan 4FSRC, Kagoshima Univ., Kagoshima, Japan

Pronuclear injection (PI) is the simplest and most widely used method to generate transgenic (Tg) mice. However, it is always associated with random integration of multiple-copy transgenes that often causes unreliable transgene expression. Recently, we and another group overcame this pitfall by developing the "PI-based targeted transgenesis", which allows targeted integration of single-copy transgene into the predetermined genomic locus using Cre-loxP or PhiC31-mediated recombination system. Here, we generated a construct carrying mutant loxPs, FRTs, and attP sites as a new combination of landing-pads with the aim of widening the choice of integration system. The resulting construct was introduced into the Rosa26 locus in ES cells with C57BL/6N genetic background and a new seed mouse line was established. We confirmed that Cre-loxP and PhiC31-mediated integration worked better than FLP-FRT-mediated system, and also found that combinational use of these two recombination systems can improve integration efficiency in both in vitro and in vivo. The resulting seed mice with defined genetic background would be useful for high-throughput, cost-saving and animal-friendly generation of Tg mice in which reliable transgene expression is guaranteed.

S 138

3P014-S Examination of BDF1 for chimeric mice host embryo

1 1 2 1 1 ○Miyuki Ida ,Tsutomu Kamisako ,Takahiro Kagawa ,Tomoo Eto ,Riichi Takahashi

1Central Institute for Experimental Animals, Kawasaki, Japan 2JAC, Inc. Tokyo, Japan

Generation of chimeric mice from ES cells is required to make the gene targeted mice. We usually employ C57BL/6J embryos for generation of chimeric mice from 129 srain derived ES cells. However, the efficiency of fertilized eggs collection from C57BL/6J is low. In this study, we verified the ability of BDF1 diproid or tetraproid embryos for chimeric mice production. C57BL/6J and BDF1 strains were used in this study. Two-cell embryos were collected by superovulated femal mice after mated with male mice. Tetraproid embryos were obtained by electrofused BDF1 2-cell embryos. Vitrification of the embryos was performed according to method of Eto et al. (2014). Zona-freed 8-cell embryos were aggregated with 129 ES cells and then transferred to uterus of pseudopregnant female mice. The collection efficiency of 2-cell embryos was 3 fold higher in BDF1 than C57BL/6J. The rate of survival after vitrification, fetal development and contribution of chimeric mice were similar in both groups. The fetal devepolpemt rate of BDF1 tetraploid was the lowest among groups. However, tetraploid had the advantage that fetus completely consisted of ES derived cells. These results suggested that BDF1 is useful in generation of gene targeted mice.

3P015-T Eradication Strategy of Murine Norovirus from SPF Rooms at a Large Animal Facility

○Kanako Shimizu, Yoshihiro Uno, Masaru Tajima

Institute of Experimental Animal Sciences Osaka University Medical School

In the Institute of Experimental Animal Sciences, Osaka University Medical School, MNV was detected in 17 of 31 rooms in November 2009. We attempted clean up all contaminated rooms by a three-year plan, and kept them clean till now, December 2013. We report the protocol used to derive MNV-free colonies.

S 139 3P016-S Production of SPF aging mouse model and its usefulness in the reproductive medicine research 1 1 1 2 ○Noboru Ogiso ,Satomi Takano ,Kaori Muguruma ,Kazumichi Yamaguchi

1Laboratory of Research Animals, National Center for Geriatrics and Gerontology, Aichi, Japan 2KAC Corporation, Kyoto, Japan

We had the opportunity to produce SPF aging animal model in connection with the operation of the new animal facility on September, 2012. To examine the usefulness of aging model raised in NCGG for reproductive medicine research, we performed in vitro fertilization (IVF) of mice and calculated fertility rate, birth rate and weaning rate. Additionally, we also performed IVF of the Senescence Accelerated Mouse (SAM) strains. In the present study, we report the result of these two experiments. Birth rate were calculated from a litter size. We also performed IVF and embryo transplantation of the SAM strains in a similar manner. We succeeded in production of 60 strains of SPF mice. The fertility rate, birth rate and weaning rate were 65.6 - 97.7%, 0.0 - 74.1% and 0.0 - 100.0%, respectively. The aging of male might have no effect on fertilizing or developmental capacity. In the case of the SAM strains, there were some differences in results among the strains. Especially in P10, birth rate showed the lowest value (11.1%) and no mouse was weaned. In the future, we plan to exam the effect of environment in culture (such as cultural composition) or the effect of gene expression related to cell cycle or cell proliferation, focusing on the SAM strains.

3P017-S Improvement of the tolerance to cryopreservation of zebrafish oocytes by enhancing AQP3-expression 1 1 1 ○Keisuke Edashige ,Momoko Hamasaki ,Masataka Takeshita ,Mizuho Kitayama1,Takeshi Hirakawa1,Chihiro Koshimoto2,Kazutsugu Matsukawa1,Magosaburo Kasai1 1College of Agriculture, Kochi University, Nankoku, Japan 2Frontier Science Research Center, Miyazaki University, Miyazaki, Japan

Fish oocytes have not been cryopreserved successfully, because it is difficult to prevent intracellular ice from forming. Previously, we showed in immature zebrafish oocytes that exogenous expression of aquaporin 3 (AQP3) promoted the movement of water and cryoprotectants through the plasma membrane. In the present study, we tried to increase its expression by extending the duration of culture of immature oocytes after injecting AQP3 cRNA, to increase the membrane permeability. When oocytes at the stage III were cultured in 90% LM containing hCG (pH 9.0) (90% LM) for 12-24 h, most of the oocytes did not mature after culture with 17α, 20β-dihydroxy-4-pregnen-3 one (DHP). When oocytes were cultured in 90% LM added with 10 nM insulin-like growth factor (IGF) for 12-24 h, about 40% of the oocytes matured after culture with DHP. When AQP3 cRNA was injected into oocytes and cultured in 90% LM containing 10 nM IGF for 12-24 h, the permeability to water and propylene glycol increased significantly. Many of the oocytes were fertilized after being matured with DHP, but they did not develop to hatching. Thus, extended culture of immature oocytes after injecting AQP3 cRNA can increase their membrane permeability. Further studies are needed to improve the culture system for supporting the developmental ability.

S 140 3P018-S Establishment of quail ES like cells

1 2 1 ○Mikiharu Nakano ,Hiroyuki Horiuchi ,Yoichi Matsuda 1ABRC, Nagoya Univ., Nagoya, Japan 2Hiroshima Univ., Higashi-Hiroshima, Japan

In mice, ES cells have been used as a powerful tool for creating transgenic models. In aves, chicken ESCs (cESCs) have been isolated from cultures of chicken blastodermal cells taken from stage X embryo. Several studies have demonstrated that cESCs can differentiate into many different types of cells in vitro and in vivo. However, because almost cESCs cannot maintain their germline competency after long-term culture, cESCs are currently unavailable for creating genetically modified chickens. We successfully established novel germline-competent cESC lines using chicken leukemia inhibitory factor (chLIF). However, no transgenic chickens still have been created using the novel cESCs due to a low efficiency of their germline transmission. Chickens have several disadvantages for performing a progeny test to examine the efficiency of germline transmission of ES cells because it takes approximately 6 months for sexual maturity of chimeric chickens. Therefore, we have selected the Japanese quail as a model animal to shorten a period of progeny test of chimeras on the following advantages: low maintenance expense and short generation time. We recently established quail ES like (qESL) cells from quail blastodermal cells cultured with chLIF. These qESL cells have the capacity for long-term successive subculture and show a competency for chimeric formation after injection into recipient embryos. We are currently examining the commitment of qESL cells to the germline to create the transgenic quails derived from qESL cells.

3P019-S Anaylsis of naive-like convert the primed-state rabbit ES cells 1 2,3 3 ○KIMIKO HONSHO ,ARATA HONDA ,Michiko Hirose ,Masanori Hatori 3,Yasmin Lubna4,Sumie Togayachi 3,Hiroyuki Miyoshi 3,Tadashi Sankai 4, Atsuo Ogura3 1Medicine and Veterinary Medicine, University of Miyazaki, Miyazaki, Japan 2Organization for Promotion of Tenure Track, University of Miyazaki, Miyazaki, Japan 3RIKEN BioResource Center,Tsukuba, Ibaraki, Japan 4Primate Research Center, National Institute of Biomedical Innovation, Tsukuba, Ibaraki, Japan

Pluripotent stem cells (PSCs) are classified as naive-state that is established from a mouse or rat and as primed-state that is established from non rodent mammalian species. We have showed that the in vitro neural differentiation capacity of the rabbit induced pluripotent stem (iPS) cells were lower than that of Embryonic stem (ES) cells, however, naive-like-state iPS cells increased more than that of the primed-state ES cells. In this study, we have analyzed whether the differentiation ability of naive-like ES cells were improved. Naive-like-state rabbit ES cells showed domed shape colonies and maintained/passaged without FGF2. Several characteristics with the formation of teratomas were confirmed. Next, we assessed the naive-like ES cells using in vitro neuronal differentiation system. As the results, the differentiation capacity of the naive-like-state ES cells increased more than that of naive-like-state iPS cells. Although naive-like ES cells efficiently contributed to the Inner Cell Mass of blastocysts, no chemric pup was obtained. These results suggested that naive-like conversion in this system couldn't improve rabbit PSCs as true naive-state.

S 141 3P020-S Comparison of the characteristics of bone marrow mesenchymal stem cells (MSCs) from gerbil and rat 1 1 2 3 ○Yui Osaka ,Masahiko Fujisawa ,Takumi Teratani ,Motoyo Maruyama ,Yoji Hakamata1 1Nippon Veterinary and Life Science University, Tokyo, Japan 2Jichi Medical University, Tochigi, Japan 3Nippon Medical school, Tokyo, Japan

MSCs are a candidate source of stem cells for regenerative therapy. However, further research is necessary to establish clinical treatment. Although MSCs have been isolated from various animals such as rats, mice, and humans, few studies are available on MSCs from the gerbil. We compared the characteristics of gerbil and rat MSCs to isolate gerbil MSCs according to the methods established for the rat. We compared the morphological characteristics of MSCs from each animal model, and gene expression profiles of specific markers of MSCs by RT-PCR, including CD29, CD105, vimentin, and collagen Iα as positive markers and CD31 and CD34 as negative markers. To test the differentiation of MSCs to adipocytes, the culture medium was added with adipogenic reagents, and cells were stained using Oil Red O. Rat MSCs were large and square in shape, whereas those from the gerbil were small and streamlined in shape. The gene expression profiles of gerbil MSCs corresponded to those of rat MSCs, except for that of CD29. Gerbil MSCs exhibited potential to differentiate into adipocytes, but the rate was much slower than that of rat MSCs. Our data demonstrated that gerbil MSCs can be eventually isolated from gerbil bone marrow using the methods established for the rat, and that the characteristics of gerbil MSCs are similar to those of other animals such as rats.

3P021-S Utility of vaginal wall impedance to determine estrus cycle phase in gerbils

○Kana Arai,Yui Osaka,Masahiko Fujisawa,Yoji Hakamata

Nippon Veterinary and Life Science University

Microscopic evaluation of a vaginal smear is the standard method for determining estrus cycle phase in animals. This method requires time and skill to obtain an accurate result. Therefore, instead of this method vaginal impedance measurements have been used to determine the estrus cycle in rats. The impedance increases only during proestrus and decreases during later phases. We evaluated the impedance measurements utility in gerbils to determine estrus cycle. The vaginal smear and impedance were examined at 12:00 pm daily for more than 2 months in both rats and gerbils, and the estrus cycle interval indicated by vaginal smear was compared with that indicated by the impedance. Results showed that the estrus cycle in the rats was divided into 4 stages and repeated stably. The impedance values during proestrus (4.1 ± 1.4 kΩ) were significantly higher than those during other phases (1.5 ± 0.4 kΩ), and the interval of each peak of impedance completely corresponded with that of smear cycle. The interval between peaks according to vaginal smear in gerbil was 5.3 ± 0.5 days. However, unlike in rats, the impedance in gerbils increased during estrus, rather than during proestrus. The impedance during estrus (12.5 ± 2.6 kΩ) was significantly higher than those during other stages (1.5 ± 0.2 kΩ). The interval between peaks according to the impedance was 5.5 ± 0.7 days, which was equal to that determined by vaginal cytology. The vaginal impedance is a very useful tool for determining the estrus cycle in gerbils.

S 142 3P022-T Effects of ovarian stimulation in Japanese macaques 1 2 2 2 ○Asako Nobukiyo ,Yoriko Indo ,Akihisa Kaneko ,Akiyo Ishigami ,Atsushi Yamanaka2,Teruhiko Hatakeyama3,Miyuki Yoshioka1,Munehiro Okamoto2,Yusuke Sotomaru1 1Natural Science Center for Basic Research and Development (N-BARD), Hiroshima University, Hiroshima, Japan 2Primate Research Institute, Kyoto University, Inuyama, Japan 3Technical Center, Hiroshima University, Hiroshima, Japan

Effects of ovarian stimulation in Japanese macaquesA Nobukiyo1), Y Indo2), A Kaneko2), A Ishigami2), A Yamanaka2), T Hatakeyama3), M Yoshioka1), M Okamoto2), Y Sotomaru1)1) N-BARD, Hiroshima Univ. 2) Primate Research Institute, Kyoto Univ. 3) Technical Center, Hiroshima Univ.Ovarian stimulation was studied to establish a foundation for reproductive technology in Japanese macaques. Mature female macaques were administered a single dose of a gonadotropin-releasing hormone agonist, continuous doses of follicle-stimulating hormone for 7 days, and a single dose of human chorionic gonadotropin after determining the menstrual cycle phase based on menstruation status and measurement of blood hormone levels. Two days after completing treatment, the ovaries were surgically exposed, follicular oocytes were aspirated, and the quantity and stages of the collected oocytes were recorded. An average of 26.0 (range, 7-73) oocytes were collected from 8 of the 9 macaques, and 28.8% (range, 23.3-42.9%) of the oocytes were mature. In addition, macaques in the fertile phase of their ovulatory cycle produced more oocytes.

3P023-S In vitro oocyte fertilization in Japanese macaques

1 1 2 ○Yusuke Sotomaru ,Asako Nobukiyo ,Teruhiko Hatakeyama ,Miyuki Yoshioka1,Yoriko Indo3,Akihisa Kaneko3,Noboru Takaesu4,Yojiro Yanagawa4,Masashi Nagano4,Munehiro Okamoto3 1Natural Science Center for Basic Research and Development, Hiroshima University, Hirshima, Japan 2Technical Center, Hiroshima University, Hirshima, Japan 3Primate Research Institute, Kyoto University, Inuyama, Japan 4Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan

Methods for in vitro fertilization of mature oocytes were studied to establish a foundation for reproductive technology in Japanese macaques. Follicular oocytes were collected from mature female macaques that had undergone hormonal ovarian stimulation, and some of these oocytes were subjected to in vitro maturation. Development of the collected mature oocytes was observed in in vitro culture after performing in vitro fertilization and intra-cytoplasmic sperm injection. Although no fertilization was observed with ejaculated sperm during in vitro fertilization, the fertilization rate with epididymal sperm was 10.0%. Injection of intra-cytoplasmic sperm resulted in fertilization of 65.5% of the oocytes and a survival rate of 85.3%. In addition, 10.0% of the eggs fertilized by in vitro fertilization and 26.3% of those fertilized by intra-cytoplasmic sperm injection developed to at least the morula stage.

S 143 3P024-S Analysis of labeling method in cell-tracking system using MSC of cynomolgus monkey 1 2 1 3 ○Yasuyo Fujishiro ,Hiroshi Koie ,Hiroaki Shibata ,Sachi Okabayashi ,Yuko Katakai3,Boran Osman1,Kiichi Kanayama2,Yasuhiro Yasutomi1,Naohide Ageyama1 1Tsukuba Primate Research Center, National Institute of Biomedical Innovation, Ibaraki, Japan 2College of Bioresource Science, Nihon University, Kanagawa, Japan 3The Corporation for Production and Research of Laboratory Primates, Ibaraki, Japan

Regenerative medicine based on stem cell transplantation is used to treat various conditions such as ischemia and spinal cord injury. In basic studies using small laboratory animals, transplanted cells improve blood flow at infarcted regions, and reconstruct an injured spinal cord and provide motor ability, though some adverse event reports. We developed cell-tracking system in a nonhuman primate model to evaluate the safety and efficacy of regenerative medicine.We rechecked isolation method of MSCs cynomolgus monkey and analyzed them by FACS. Then we did time lapse visualization with fluorescent microscopy while labeling MSC with FIP to identify the process. A cynomolgus monkey was then transplanted with the FIP-labeled MSCs and assessed by MRI.We could isolate MSCs completely and were confirmed by FACS. Then we could reveal the process of labeling with FIP in MSC by fluorescent microscopy. Several organs of cynomolgus monkeys emitted MRI signals from the FIP-labeled cells. The established cell-tracking system with FIP-labeled MSC in nonhuman primates was safe and efficient rather than any other methods of regenerative medicine assessment.

3P025-S Pattern of gene expression during marmoset preimplantation embryo 1 2 1,2 ○Ayako Sedohara ,Hideyuki Okano ,Sasaki Erika

1Department of Applied developmental Biology, Central Institute for Experimental Animals, Kawasaki, Japan 2Keio University Graduate School of Medicine, Tokyo, Japan In primate, ES cells also have pluripotency observed as mouse ES cells however, do not have ability to produce chimera. Although ES cells are derived from blastocyst in mouse and primate, the morphology of the primate ES cells resemble the pluripotent cell lines derived from post-implantation mouse epiblasts. It is predicted that there are some differences of developmental mechanisms between mouse and primate. To identify the differences of development between mouse and primates, we tried immunostaining for various genes, such as Oct3/4, Nanog, Sox2, Cdx2, and Gata6 expressed in preimplantation embryos in common marmoset (Callithrix jacchus). As a result, the expression pattern of early genes in marmoset is different from the mouse reported previously. The expression of Oct3/4, Sox2, Cdx2, and Gata6 were detected at nucleus in 8-cell stage embryos. Nanog was observed in 16-cell stage embryos. Co-expression of those genes was persisted in late blastocyst; inner cell mass expressed both Cdx2 and Gata6, and outer cells expressed Oct3/4, Sox2, and Nanog. In late blastocysts hatched from zona pellucida, Oct3/4-expressing cells were separated clearly from Cdx2-expressing cells as Gata6. Oct3/4 was co-expressed with Sox2 and Nanog at very restricted region in hatched late blastocyst. Gata6-expressing cells were observed in the blastocoelic surface of hatched late blastocyst.

S 144 3P026-S Towards a INSR gene knock down transgenic model of type II diabetes in a common marmoset 1,2 3 1 1 1 ○Tsukasa Takahashi ,Takuji Maeda ,Takeshi Kuge ,Kenya Sato ,Ryoji Ito ,Erika Sasaki1 1Central Institute for Experimental Animals, Kanagawa, Japan 2Keio University School of Medicine, Tokyo, Japan 3 Nagoya University , Aichi, Japan

Recently, genetically modified primates have been generated from several kinds species of non-human primates. However, transgenic non-human primate that shows loss-of-gene function has not been reported. Currently, we are attempting to generate a target gene knock down marmoset using the vector coding the insulin receptor (INSR) - specific shRNA under tet regulator and ubiquitous EGFP expression as integration marker. In this vector the Dox treatment leads the siRNA and ablation of INSR protein results in onset of type II diabetes. Six shRNA were designed from marmoset INSR cDNA sequence and Western blot analysis were performed to evaluate RNAi efficiency of these shRNA. The highest efficient shRNA lentiviral vector no.3 was injected into marmoset embryos and the GFP expressing embryos were transferred to surrogate mothers non-surgically. The highest efficient shRNA expressed vector led to 73% reduction of INSR expression. The shRNA expression lentiviral vector showed high transduction efficiency more than 90% in embryos and two transgenic offspring were successfully derived. Although these offspring were dead on the day 4 and day 5 after birth, respectively, expression of GFP and knockdown of INSR were confirmed in the bone marrow, liver cells and fibroblast which we collected from dead offspring. The embryo transfer is continued and two recipients are pregnant.

3P027-S Genetic control and constant production of MMP by somatic cell cloning 1 1 1 2 3 3 4 ○M Shibata ,S Enya ,M Otake ,T Kawarasaki ,S Mikawa ,H Uenishi ,T Nishimura

1Shizuoka Prefectural Swine and Poultry Research Center,Shizuoka,Japan 2Tokai Univ. 3NIAS 4Fuji Micra Inc.

Fuji Micra Inc. introduced a small pig for research called Microminipig (MMP) . MMPs weigh around 10kg at six to seven months of age. Current study was to reveal the data of growth, coat color, types of swine leukoantigen (SLA) etc. and to establish an efficient method for producing MMPs using somatic cell cloning and gene analysis. 1) Growth and coat color of 73 MMPs were examined. SLA types of 69 MMPs were analyzed by using 11 microsatellite markers. Coat color-related gene was analyzed by determining the allele of KIT gene from 24 white MMPs. 2) Commercial MMPs with white coat and homozygous SLA type were produced. A doner for somatic cell cloning was selected from the population to make grandparents based on the data collected. Parents and commercial MMPs were produced by natural mating. 1) The average weight of six month-old MMPs was 9.4kg (n=15) in male, 9.0kg (n=19) in female. Nine SLA haplotypes were found. The coat colors were white, grey and black. One white MMP was homozygote of the dominant white color allele. 2) Among grandparents, grandfathers were selected by SLA types and coat color-related gene and grandmothers were by SLA types and productivity. Eighteen grandparents mated naturally and produced one father, 19 mothers and 41 commercial MMPs. Commercial MMPs had homozygous SLA type with white coat. Males weighed 8.9kg (n=12) on average at six months old and females 9.9kg (n=7).

S 145 3P028-S Production protocol and characterization of NOG-hairless mouse 1 1 1,2 1 ○Yuyo Ka ,Tomoyuki Ogura ,Takuma Mizusawa ,Tsutomu Kamisako ,Riichi Takahashi1,Kazuaki Ito1,3,Hideki Tsutsumi1,Tomoo Etoh1,Mamoru Ito1,Kyoji Hioki1 1Central Institute for Experimental Animals,Kawasaki,Japan 2JAC Inc,Tokyo,Japan 3WORLD INTEC,

NOG-hairless mouse developed in our laboratory is developed for the purpose of practical use for PDX (Patient Derived Xeno-transplantation) model . Its hairless character is effective to observe tumor growth and metastasis in bio-imaging research. In this report, we established standard pyramid management plan (Foundation/Expansion/Production colony). In the Production colony, breeders are continually housed as pairs of one or two females and one male from 10 to 32 week old (or up to the 4th delivery). hr/hr and hr/+ gene was judged in the state of hair. Body weight was measured once in a week from 8 to 23 week old, and main outward appearance was observed from 8 to 23 week old. The average reproduction performances obtained from 105 matings of PC is; fertility rate is 33%; average litter size is 7.6; weaning rate is 92.3%. We could obtain 329 NOG-hr/hr mice (157 females, 172 males) and 666 NOG-hr/+ mice (333 females, 333 males). Only male NOG-hr/hr mouse tend to delay weight gain compared with male NOG-hr/+ mouse. The hairless rate increased gradually in both female and male NOG-hr/hr mice, repeating depilation and growth of downy hair (about 1 cm). The character of ingrown toenail was observed with all the NOG-hr/hr mice.

3P029-S Production protocol and characterization of BRG-nude mouse 1,2 1 1 1 ○Takuma Mizusawa ,Tomoyuki Ogura ,Yuyo Ka ,Tutomu Kamisako ,Riichi Takahashi1,Tomoo Etoh 1,Mamoru Ito1,Kyoji Hioki1 1Central Institute for Experimental Animals,Kawasaki,Japan 2JAC Inc,Tokyo,Japan

BRG-nude mouse was introduced Foxn1 nu (nu/nu) mutation into the severe immunodeficient BRG mouse (BALB/cAJic.Cg-Rag2tm1Fwa Il2rgtm1Sug/Jic). A nude mouse has been used in the subcutaneous inoculation of a human tumor cell lines for many years, and it is thought that a hairless character is siginificant for bio-imaging research field. Bio-imaging enables to observe tumor growth and metastasis using live mice. In this report, we established standard pyramid management plan (Foundation/Expansion/Production colony). In the Production Colony, breeders are continually housed as pairs of one or two female with one male from 12 to 32 weeks old (or up to the 4th delivery). The nu/nu or nu/+ gene was judged by their hair. Body weight was measured once in a week from 5 to 20 weeks old and general internal organ weight and a blood characteristic were analyzed at 20 weeks old. We confirm the reproduction performance under these breeding conditions.The reproduction performance obtained from 150 matings of PC is; fertility rate is 58.2%; average litter size is 4.0; weaning rate is 82.5%. We could obtain 442 BRG-nu/nu mice (225 females, 217 males) and 687 BRG-nu/+ mice (369 females, 318 males). Body weight gain of BRG-nu/nu was relatively smaller compared with BRG-nu/+. Other characteristic data is also reported.

S 146 3P030-S Production protocol and characterization of TK-NOG mouse 1,2 1 1 1 ○Kayo Tomiyama ,Tomoyuki Ogura ,Yuyo Ka ,Tsutomu Kamisako ,Riichi Takahashi1,Mamoru Ito1,Kyoji Hioki1,Hiroshi Suemizu1 1Central Institute for Experimental Animals,Kawasaki,Japan 2JAC Inc,Tokyo,Japan

TK-NOG[NOG-Tg(ALB-UL23) 7-2/ShiJic] based on severe immunodeficient NOG® mouse background can introduce Xeno-Hepatocytes. It can generate "humanized mouse model" which is useful for drug development for human hepatitis virus, and drug metabolism examination by transplanting a human normal hepatocyte. Since TK-NOG strain is sterility in male under the influence of Tymidin Kinase gene, it is maintained as heterozygous TK-NOG Tg femal x Non-Tg male siblings. We established standard pyramid management plan (Foundation/Expansion/Production colony). For producion colony, we prepared 20 of male mice and 140 of female mice divided into 7 groups. Breeders are housed one week and rotating serial mating from 12 to 32 weeks old(or up to the 4th delivery). Transgene screeing was carried out by the PCR using tail tip taken at 4-week old. The average reproduction performance in this serial mating; fertility rate is 77.5%; average litter size is 6.0; weaning rate is 94.9%. We could obtain about 3145 TK-NOG mice (1580 females, 1565 males) and 3166 Non-Tg mice (1558 females, 1608 males). About 20 TK-NOG males mice which are the targets of a liver transplant can produce every week. From this index, we are setting up a system to boost our production capacity. Unless hepatic insufficiency is induced by gancicrovile, there is no difference of weight gain between TK-NOG and Non-Tg litter mate. Other characteristic data is also reported.

3P031-S Ovulation phase of cynomolgus monkeys estimated by measurement of serum estradiol 1 2 2 2 ○Nobuhiro Shimozawa ,Mutsumi Togo ,Yoshimi Otsu ,Hayato Narita ,Yasuhiro Yasutomi1 1Tsukuba Primate Research Center, National Institute of Biomedical Innovation, Tsukuba, Japan 2The Corporation for Production and Research of Laboratory Primates, Tsukuba, Japan

In conventional breeding systems, one female cynomolgus monkey is typically housed together with one male for 72 hr on and after the anticipated day of ovulation, which is the period from the 11th to the 14th day of the menstrual cycle. This system can contain multiparous and nulliparous females that do not become pregnant for a long time. In order to clarify whether females ovulate for the period from day 11 to 14 after menstruation, we examined the ovulation phases of nulliparous cynomolgus monkeys at four to six years of age by measuring serum estradiol using an automated immunoassay analyzer. The day following the peak in serum estradiol is generally considered to be the day of ovulation. We estimated that ovulation occurred at a rate of approximately 40% of examined monkeys in each of day 11 to 14 and day 15 to 22 after menstruation. It was thus estimated that, in conventional breeding systems, the proportion of one female and one male that are housed together during the actual period of ovulation is lower than half. This may be responsible for females not becoming pregnant for a long time. In order to breed cynomolgus monkeys efficiently, it may be necessary to change the period when pairs are housed together.

S 147 3P032-S Produce of SPF Akita big rabbit

1 1 1 1 2 ○Yukihisa Matsuda ,Tamako Ikeda ,Yoshiko Shibata ,Keita Basaki ,Yuko Tsuya 1Animal Research Laboratory, Bioscience Education-Research Center, Akita University, Akita, Japan 2JAC,Ltd. tokyo, Japan

Introduction:In Senpoku region of Akita prefecture, bigger rabbits are reared. The body weight of them are around 7kg. We previously obtained SPF big rabbits by caesarian section and foster mother method. However we couldn't maintain them because of reproduction disturbance by inbreeding depression. We started again to produce SPF big rabbits. In this presentation we introduce the growth curve of SPF big rabbits. Methods:As foster mothers and recipients of embryo transfer method, SPF female KBL (KITAYAMA LABES) were used. Rabbits were fed freely R Stock (NOSAN). Cages size were 653x653x450mm (TECNPLAST). In this experiment embryo transfer method and caesarian section were carried out. In order to produce SPF big rabbits several male and female big rabbits were obtained from farm. About 10 embryos were transferred 13 respectively recipients. Results and Discussions:20 new born were obtained from 13 dam, but only 2 new born weaned and now exists. 4 new born obtained from farm dam applied caesarian section. These 3 males and 3 females were examined about growth curve. 8 weeks after big rabbits of both males and females were bigger than those of KBL. At 36 weeks body weight of male big rabbits, female big rabbits, male KBL, and female KBL were 5,230±186g, 6,609±408g, 3,418±302g, and4,124±339g, respectively. For preventing from in breeding depression, used with sperm of farm big male rabbits artificial insemination are conducted for SPF female big rabbits.

3P033-S Search for genes responsible for the stress-induced hypertension in SHRSP

○Toru Nabika,Hiroki Ohara,Minoru Isomura,Kaoru Niiya

Deprtment of Functional Pathology, Shimane University School of Medicine

The sympathetic nervous system (SNS) plays an important role in regulating the blood pressure (BP) in response to various environmental stimuli. The stroke-prone spontaneously hypertensive rat (SHRSP) is well known to have an exaggerated sympathetic response to stress, which may be causally related to its severe hypertension. Recently, based on the results of physiological analysis of congenic strains established between SHRSP and normotensive Wistar-kyoto (WKY) rat, we revealed that a gene (or genes) responsible for exaggerated sympathetic response to stress in SHRSP was located in a 1.2-Mbp fragment on chromosome (Chr) 1. In this study, we explored genes responsible for exaggerated stress response in SHRSP in this chromosomal fragment. Gene expression analysis on the genes located in the target region showed that no significant differences of the expression between SHRSP and the normotensive control, WKY. On the other hand, analysis of coding sequences of the genes revealed that a gene harbored a nonsense mutation. Additional sequencing of 19 different rat strains indicated that this mutation was specific for substrains of SHRSP. Although the function of the genes in the regulation of blood pressure was not clarified, Stim 1 was considered a promising candidate gene for the exaggerated stress response in SHRSP.

S 148 3P034-S Prolongation of anagen in hair cycle and a 1-bp deletion of Fgf5 in long-haired coat Syrian hamster 1 1,2 1 ○Yasuhiro YOSHIZAWA ,Kenta WADA ,Gaku SHIMOI ,Yuichi KAMEYAMA1,Katsuhiro FUKUTA3,Ryoichi HASHIZUME1 1Graduate School of Bioindustry, Tokyo University of Agriculture, Japan 2Mammalian Genetics Project, Tokyo Metropolitan Institute of Medical Science, Japan 3Tsukuba Primate Research Center, National Institute of Biomedical Innovation, Japan

PURPOSE: MALC is a strain of Syrian hamster, exhibits long hair transmitted via autosomal recessive inheritance (Fukuta et al. 1993). We investigated hair cycle in MALC and wild-type (WT), and attempted to identify the responsible mutation of the genes causing long-hair in MALC.Materials and methods: To compare the hair cycle between MALC and WT, hamsters were depilated at 12 weeks old. After depilation, skin tissues were harvested for preparing sections. The coding sequences of Fgf5 were determined by RT-PCR using primer sets designed in highly conserved region.RESULTS: Histological differences between MALC and WT were not observed in skins at 1st and 15th day after depilation. At 30th day after depilation, the hair follicles (HFs) regression was found in WT. However, that of MALC showed growth phase of HFs. Delaying of catagen induction was shown in MALC. Our functional cloning strategy for malc discovered a deletion of 1-bp at 546th nucleotide position in Fgf5 cDNA in MALC. This deletion might be led to truncation after 183rd amino acid residues resulting from frame shift in FGF5 of MALC. This mutation is in highly conserved region through evolutional history. Thus, the mutation of Fgf5 was detected in MALC might be a causing of long hair.

3P035-S Phenotype analysis of femal of ENU induced mutant mouse with infertility

○Mizuho Chikushi,Yasuhiro Fujiwara,Hirokazu Matsumoto, Kouyou Akiyama, Takehito Tsuji,Tetsuo Kunieda Okayama University, Graduate School of Environmental and Life Sience, Okayama, Japan

The repro57 is an ENU induced mutant mouse showing arrest of spermatogenesis in male, but the phenotype of repro57 female has not yet characterized. Since repro57 female never get pregnancy after mating with normal males, repro57 female was suggested to be infertile. To reveal the cause for the infertility, we analyzed the reproductive traits of repro57 females including follicles growt, ovulation, and early embryonic development. As a result of histological examination of ovaries, all stages of follicle and corpus luteum were observed in repro57, and there was no difference between wild type and repro57 mutant. However, when ovulated oocytes werw cultuerd for 5 days, the oocytes from repro57 female did not develop beyond the 4-cell stage, while those of the wild type female developed until blastocyst stage. Thus the cause of the infertility in repro57 was suggested to be the arrest of development at early embryonic stage. The infertility of the repro57 male is due to the arrest of spermatogenesis caused by defective meiosis. Therefore, the abnormality in the embryonic development of the repro57 female is suggested to be caused by the defect of meiotic.

S 149 3P036-S Development of NGS based DNA typing method of the MHC genes in common marmoset 1 1 2 3 ○Takashi Shiina ,Atsuko Shigenari ,Shuya Mori ,Kazutaka Kitaura ,Yoshie Kametani1,Ryuji Suzuki3

1Department of Molecular Life Science, Tokai University School of Medicine, Kanagawa, Japan 2Department of Hematology, Tokai University School of Medicine, Kanagawa, Japan 3Department of Rheumatology and Clinical Immunology, Clinical Research Center for Allergy and Rheumatology, Kanagawa, Japan

The common marmoset (Callithrix jacchus) is a New World monkey that has emerged as an important experimental animal model for biomedical research in various domains (transplantation of iPS differentiated cells, response to drugs and sensitivity to experimental diseases). However, detailed knowledge concerning to the major histocompatibility complex (marmoset MHC; Caja) is still lacking. From our previous studies, genomic sequences of 1,933 kb in total have been determined that include nine Caja-B genes, 14 Caja-G genes and 5 Caja-F genes. Here, we report development of a new DNA typing method of Caja genes by next generation sequencing (NGS) and describe the genetic features characterized by NGS and sub-cloning methods of 45 animals. By NGS method 11 to 17 kinds of Caja class I sequences were detected per animal, and all sequences included Caja class I sequences detected by sub-cloning method. From the polymorphism information at least 10 kinds of Caja-G haplotypes were estimated, and 2 to 7 transcribed Caja-G genes were detected per haploid. Therefore, the NGS based DNA typing method provide precise allele and haplotype data for biomedical research such as transplantation studies and genetic diversity studies.

3P037-S Quantitative comparison between SRY and DYS14 in detecting cell free fetal DNA of pregnant monkeys

○Lubna Yasmin,Jun-ichiro Takano,Tadashi Sankai

Tsukuba Primate Research Center, National Institute of Biomedical Innovation

Cell-free fetal DNA in maternal serum can be useful for detecting prenatal disorders and pregnancy monitoring. More sensitive, specific and quantitative detection of cell-free fetal DNA in maternal serum may expand the several utility of such measurement. Improvement might be made by amplifying Y-specific sequences that are highly repetitive within the Y chromosome like DYS14 sequence located in the TSPY (testis-specific protein, Y-linked). We have optimised a protocol for the multiplex qPCR amplification for the multi copy sequences DYS14 and single-copy SRY gene. PCR detection was performed by using plasma DNA extracted from pregnant cynomolguls monkeys. These DYS14 and SRY quantifications were compared with the total cell-free DNA quantified targeting the genomic region of the single-copy gene of the chromosome, NQO1 (NADPH: quinone oxidoreductase). Absolute copy number for NQO1, SRY and DYS14 were successfully determined. The mean absolute copy numbers for total cell free DNA were 2.24×104 copies per mL plasma. The multi copy DYS14 like sequences detection in cell free DNA is at least 10 fold more sensitivity than the single copy SRY assy. Moreover DYS14 assay was able to detect increased copies of fetal DNA per PCR reaction compare to SRY. In cell free fetal DNA, DYS14 could be able to detect easily while SRY showed limited copies. DYS14 assay is the improvements for quantifying cell free fetal DNA in maternal plasma of cynomolgus monkeys.

S 150

3P038-T Development of a NGS based DNA typing method of MHC genes for Cynomolgus Macaque 1 2 3 3 ○Yukiho Yamada , Keiko Tanaka , Chizuru Iwatani , Hideaki Tsuchiya , Yasushi Itoh4, Kimio Yoneda1, Hisashi Yamanaka1, Hiroshi Nakagawa1, Masao Ota5, Hidetoshi Inoko2, Kazumasa Ogasawara4, Takashi Shiina2 1Ina Research Incorporated, Nagano, Japan 2Department of Molecular Life Science, Tokai University School of Medicine, Kanagawa, Japan 3Research Center for Animal Life Science, Shiga University of Medical Science, Shiga, Japan 4Department of Pathology, Shiga University of Medical Science, Shiga, Japan 5Department of Legal Medicine, Shinshu University School of Medicine, Nagano, Japan The purpose of this study was to develop DNA typing methods for Cynomolgus Macaque MHC gene using next generation sequencers. As a result, it was shown that a combination of serial multiplex PCR and next generation sequencers took advantage of providing quick and simple methods, and would accelerate transplantation studies using Cynomolgus.

3P039-S Improved high-throughput gene profiling system detailed detectable SNPs among B6 substrains

○Ikuo Miura,Akiko Shinogi,Daiki Usuda,Tomohiro Suzuki,Hideki Kaneda, Tamio Furuse, Kimio Kobayashi,Ikuko Yamada,Masaru Tamura,Hiroshi Masuya, Shigeharu Wakana RIKEN BioResource Center, Tsukuba, Japan

Aim: In performing phenotype analysis for genetically engineered (GE) mouse strains, it was considered that there was affection of ununiformed genetic background arose by crossing of different inbred strains. High-throughput gene profiling system constructed in the Japan Mouse Clinic was consisting 192 marker set based on TaqMan assay and utilizing for the evaluation of uniform genetic background for the GE strains. This system was possible to get information such as the ratio of replacement to backcrossing strain, the paternal or maternal origin and the existence of transgenes as gene modification and, moreover, was able to closely distinguish each C57BL/6 (B6) substrains and production breeders (PBs) of B6/J by SNPs markers. However, it was not containing SNPs to detect PBs of B6/N, because SNPs among B6/N were not available on public databases (DB) in those days. We find new SNPs among B6/N from updated public DB and improve this system by adding those SNPs markers. Method: About 150 SNPs among B6 strains were selected from public DB and check allele pattern with seven PBs from B6/J and B6/N by DNA sequencing. New recognized SNPs were added as marker to detect B6/N. Conclusion: We found 13 SNPs among PBs of B6/N and SNPs between B6/J and B6/N on all autosomal chromosomes. The improved system by adding these SNPs markers becomes detectable in more detailed genetic background.

S 151 3P040-S Optimizing transfection condition of TALEN in porcine fetal fibroblasts 1 2 2 1 ○Shiro Yamashita ,Tetsushi Sakuma ,Takashi Yamamoto ,Yutaka Sendai

1ZENNOH Central Research Institute for Feed and Livestock, Ibaraki Japan 2Graduate School of Science, Hiroshima University, Hiroshima Japan

In order to analyze the gene function of livestock, we are producing gene knockout (KO) livestock. To produce KO livestock, we generally establish KO cell lines by transfecting KO vector to fetal fibroblasts and perform the somatic cell nuclear transfer. However, the establishment efficiency of KO cell lines is low. Recently, it was reported that artificial nuclease such as ZFN, TALEN, and CRISPR/Cas9 can perform genetic modification (including KO) very efficiently in various cells. In this study, we optimized the transfection conditions of the Platinum TALEN expression vector and mRNA in porcine fetal fibroblasts.As a result of examining the optimal introducing method, it became clear that expression vectors could be introduced into high rate by the nucleofection (amaxa) and that mRNAs were by the lipofection (Lipofectamine2000). After introducing TALEN expression vectors (2 µg), the mutation rates when cultivating by three kinds of culture conditions(3 d at 38.5°C, 1 d at 38.5 °C followed by 2 d at 30°C, or 3 d at 30°C) was investigated, it became 5.2%, 6.2% and 8.3%, respectively. In case of mRNAs (2 µg), mutation rates were 2.8%, 12.7% and13.3%, respectively. In addition, when the expression vectors were used, integration of the vector into a genome was confirmed. Thus, we concluded that mRNA transfection is the most suitable method for genetic modification of porcine fetal fibroblasts.

3P041-S Phosphorylation of Angiomotin regulates the Hippo pathway in preimplantation mouse embryos 1,2 3 3 4 ○Yoshikazu Hirate ,Shino Hirahara ,Ken-ichi Inoue ,Atsushi Suzuki ,Vernadeth Alarcon5,Kazunori Akimoto4,Takaaki Hirai6,Kazuhiro Chida6,Shigeo Ohno4,Yusuke Marikawa5,Kazuki Nakao3,Akihiko Shimono7,Hiroshi Sasaki2 1Center for Experimental Animal, Tokyo Medical and Dental University, Tokyo, Japan 2IMEG, Kumamoto Univ., Kumamoto, Japan 3CDB, RIKEN, Kobe, Japan 4Yokohama City Univ., Yokohama, Japan 5Univ. of Hawaii, Honolulu, USA 6Univ. of Tokyo, Tokyo, Japan 7TransGenic, Kobe, Japan

Purpose: In preimplantation mouse embryos, the outer and inner cells are specified to the trophectoderm and inner cell mass, respectively. The inner cell mass is induced by inner cell-specific activation of the Hippo pathway. The roles of a junctional scaffold protein Angiomotin (Amot) and cell polarity in Hippo signaling is investigated.Materials and methods: Amot and cell polarity genes are manipulated to examine their roles in Hippo signaling in preimplantation mouse embryos. Molecular interactions and actin-binding of Amot are also examined in cultured cells.Results: Amot is essential for Hippo pathway activation. In the nonpolar inner cells, Amot localizes to adherens junctions (AJs). In the outer cells, the cell polarity sequesters Amot from basolateral AJs to apical domains, suppressing Hippo signaling. The N-terminal domain of Amot is required for interaction with E-cadherin, Lats kinase and F-actin. In AJs, S176 in the N-terminal domain of Amot is phosphorylated by Lats, which inhibits the actin-binding, thereby stabilizing the Amot-Lats interaction to activate the Hippo pathway.

S 152 3P042-T Downsized experimental beagle (TOYO beagle) and comparison of background data 1 1 1 1 ○Yoichi Kinoshita , Yuichiro Harada , Shingo Harada , Katsuzi Kuba , Hiroshi Morita1, Takehiro Aihara1, Yoshibumi Kuwabara2 1Kitayama Labes Co., Ltd. 2Oriental Yeast Co., Ltd.

By changing breeding protocol to select small skeleton and lightweight animals, TOYO Beagle returned to adult size of 9-10 kg, comparable to those of 1980's. Feeding was good. In the period of group housing food consumption was improved by keeping the number of individuals in a group constant, and by separation of males and females. There was no particular change in the background data of rearing dogs.

3P043-S PlGF plays a critical role in fetus development

○Jin Hee Lee, Jae Woong Ryoo

Kyungpook national university (South of Korea)

Angiogenesis is an important biological process during development, reproduction and in immune responses. Placental growth factor (PlGF) is a member of vascular endothelial growth factor (VEGF) that is critical for angiogenesis and vasculogenesis. We generated transgenic mice over-expressing PlGF in specifically T-cells using the human CD2 promoter to investigate the effects of PlGF over-expression. Transgenic mice were difficult to obtain owing to high lethality; for this reason, we investigated why gestational loss occurred in these transgenic mice. Here we report that placenta detachment and inhibition of angiogenesis occurred in PlGF transgenic mice during the gestational period. Moreover, even when transgenic mice were born, their growth was restricted. Conclusively, PlGF over-expression prevents angiogenesis by inhibiting Braf, ERK activation and down-regulation of HIF-1α in the mouse placenta.

S 153 3P044-S Over-expression of Roquin aggravates T cell mediated hepatitis in transgenic mice using T cell specific promoter ○Min Jee Choi, Young Hun Sung, Jae Woong Ryoo

Kyungpook national university (South of Korea)

Chronic hepatitis is a major cause of liver cancer, so earlier treatment of hepatitis should reduce liver cancer incidence. Hepatitis can be induced in mice by treatment with Concanavalin A (Con A); the resulting liver injury causes significant CD4+ T cell activation and infiltration. In these T cells, Roquin, a ring-type E3 ubiquitin ligase, is activated. To investigate the role of Roquin, we examined Con A-induced liver injury and T cell infiltration in transgenic (Tg) mice overexpressing Roquin specifically in T cells. In Roquin Tg mice, Con A treatment caused greater increases in both the levels of liver injury enzymes and liver tissue apoptosis, as revealed by TUNEL and H&E staining, than wild type (WT) mice. Further, Roquin Tg mice respond to Con A treatment with greater increases in the T cell population, particularly Th17 cells, though Treg cell counts are lower. Roquin overexpression also enhances increases in pro-inflammatory cytokines, including IFN- γ, TNF- α and IL-6, upon liver injury. Furthermore, Roquin regulates the immune response and apoptosis in Con A induced hepatitis via STATs, Bax and Bcl2. These findings suggest that over-expression of Roquin exacerbates T-cell mediated hepatitis.

3P045-S Jazf1 overexpression regulates lipid metabolism in HFD

○Hye Rim Kim, Wook Bong Kwon, Jae woong Ryoo

Kyungpook national university (South of Korea)

JAZF1 is a 27 kDa nuclear protein containing two putative zinc finger motifs that is associated with diabetes mellitus and prostate cancer according to genomewide association studies; however, little is known about the function of this gene in regulating metabolism. Recent evidence indicates that JAZF1 transcription factors bind to the nuclear orphan receptor TR4 and act as a strong repressor. This receptor regulates PEPCK, the key enzyme in gluconeogenesis, at the transcriptional level. Excess PEPCK expression in mice causes hyperglycemia, hyperinsulinemia, and increased glucose turnover. Therefore, we hypothesized that ectopic expression of Jazf1 may lead to abnormal expression of PEPCK that allow for the metabolic disorder. To elucidate its role in metabolism, we fed the mice with high- or normal- fat diet up to 18 weeks. In the liver tissue of mice, Jazf1 overexpression led to a substantial reduction in the expression of PEPCK. In Jazf1 overexpression mice, weight gain was found to be significantly decreased and increment of blood glucose level also decreased. Our data suggest that Jazf1 plays a critical role in the regulation of energy and lipid homeostasis, and promotes the development of metabolic disorder. Jazf1 may provide a new therapeutic target in the management of obesity, diabetes, and liver steatosis.

S 154 3P046-S Practical comparisons among 4 nucleic acid amplification tests for the detection of murine norovirus 1 2 3 4 5 ○Ken-Ichi Hanaki ,Fumio Ike ,Kouji Sakai ,Yasushi Ami ,Shigeru Kyuwa

1Center for In Vivo Sciences, Iwate Medical University, Iwate, Japan 2Experimental Animal Division, RIKEN BRC, Ibaraki, Japan 3Department of Virology III, National Institute of Infectious Diseases (NIID), Tokyo, Japan 4Division of Experimental Animal Research, NIID, Tokyo, Japan 5Department of Biomedical Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan

Murine norovirus (MNV) is a non-enveloped virus with a positive sense and single-stranded genomic RNA, and the most common contaminant in laboratory mouse colonies worldwide. To detect variants of MNV, broadly reactive nucleic acid amplification tests (NATs) such as nested RT-PCR (nRT-PCR) and TaqMan real-time RT-PCR (TaqMan) assays have been developed. For the purpose, we have developed RT-LAMP and SYBR Green I real-time RT-PCR (Green) assays. In this study, we compared the diagnostic performance of 4 NATs using fecal RNA specimens of mice deposited from 19 laboratories in Japan and a laboratory in US. Assays of nRT-PCR, TaqMan, RT-LAMP and Green were completed in about 360, 175, 90 and 65 min, respectively. Of 120 specimens, MNV was detected in 57 by nRT-PCR and by Green, in 54 by RT-LAMP, and in 56 by TaqMan assays. Compared with nRT-PCR assay, the other 3 assays exhibited 100% specificity. In conclusion, RT-LAMP is expected as a cost-effective and practical alternative to RT-PCR-based assays. Green assay is the most feasible method with rapidity and sensitivity, although it needs an expensive instrument.

3P047-S Genome analysis of persistently infected Hepacivirus which is related to hepatitis-C in primates 1 1 1 2 ○Atsunori Higashino ,Saori Suzuki ,Akatsuki Saito ,Kazuhiro Matsuoka ,Hirotaka Ode2,Yuko Katakai3,Sachi Okabayashi3,Ken-ichi Mori4,Noboru Maki4,Hirofumi Akari1 1Primate Research Institute, Kyoto University, Inuyama, Japan 2National Hospital Organization, Nagoya Medical Center, Nagoya, Japan 3Corporation for Production and Research of Laboratory Primates, Tsukuba, Japan 4Advanced Life Science Institute, Wako, Japan

Hepatitis C virus (HCV) has been shown to induce a variety of quasispecies due to the high mutation rate in the viral genome. The quasispecies occurs with selection bias as a result of the viral evasion of immune system. It is therefore possible that analyzing sequential dynamics in the evolution of the viral quasispecies will help understanding in detail the viral strategy for the immune evasion. However, there are several hurdles regarding the analysis, including availability of sequential clinical samples, variety of the viral genotypes and quasispecies at the viral transmission. A common marmoset model for GB Virus B (GBV-B), which as well as HCV belong to Hepacivirus genus and is closely related to HCV, seems to be promising since GBV-B infection develops persistent infection and chronic hepatitis C-like diseases. Here we analyzed sequential dynamics in the viral evolution in marmosets with different disease progression and identified a number of mutations specific for chronic hepatitis. Further analyses of the viral genome will unveil hepaciviral strategy for the viral pathogenesis and immune evasion.

S 155 3P048-S RIKEN BRC mice are free of Murine Norovirus and Murine Astrovirus

○Ayako Kajita,Chiimi Ogawa,Hiromi Sakata,Shinobu Yuuki,Atsushi Yoshiki, Fumio Ike Experimental Animal Division, RIKEN BRC, Tsukuba, Japan

Murine norovirus (MNV) is known as a major contaminant in mouse colonies in the world. Recently, murine astrovirus (MuAstV) is reported prevalently found in mouse colonies. Since RIKEN BioResource Center (BRC) performs rederivation on all deposited strains, mice housed in barrier rooms (after rederivation) should be free of both viruses. In order to clarify this assumption, we tested fecal samples of dirty-bedding sentinel mice housed in conventional (before rederivation) and barrier rooms of BRC. [Materials and methods] RT-PCR assays were conducted using MNV and MuAstV specific primers (Comp. Med. 2006, 56:247-251, PLoS ONE 2013, 8:e66937). Amplified products were sequenced for homology search. [Results] MNV and MuAstV were detected in 28 samples and 22 samples out of 42 feces obtained from sentinel mice housed in conventional rooms, respectively, suggesting that both viruses are prevalent in originally housed environments. Five samples selected from positive samples at random were sequenced. Three samples were completely conformed to MNV MT30-2, other 2 samples showed 95% and 99% homologies. All of MuAstV positive samples showed homologies of 95-96% with MuAstV STL1. Neither MNV nor MuAstV was detected in 49 sentinel mice housed in barrier area. [Conclusion] As far as we examined in this study, MNV and MuAstV were eliminated by rederivation. However we will continue to carefully monitor these agents not only by using sentinels but also by using individual mouse.

3P049-S Bacillus piliformis(Tyzzer) contamination of rats in the animal facility

○Hiroshi Yamamoto,Hironori Izumi,Hikari Minamisawa,Tadahiko Tsuchiya, Satoshi Ohtsuka, Noriko Tsuneda Division for Animal Resources and Development, Life Science Research Center,University of Toyama

Microbial monitoring is essential for the proof of the reliability of the experimental data as well as improve the quality of laboratory animals. In the animal facility of Toyama University, in microbial monitoring, contamination of Bacillus piliformis(Tyzzer) in rat room was happened at 1 or 2 rooms for several times. The blood from sentinel rats was subjected to the inspection by ELISA test by Moniraiza Kit and IFAT. When the inspection by ELISA test was positive, the sample was sent to the Central Institute for Experimental Animals to reconfirm the results. Tyzzer-positive rat was confirmed in rat room (No.1) (February, 2005), rat special maintenance room (November, 2007), rat room (No.1) (May, 2009), and rat room (No.1) and strain animal maintenance room (June, 2010). The number of the tyzzer-positive rats among sentinel was 2 rats among 4 in rat special maintenance room (2007), 2 rats among 5 in rat room (No.1)(2009) and 4 rats among 7 in rat room (No.1)and strain maintenance room (2010). The rat maintained for experiments in the same room that the sentinel was positive was also examined and Tyzzer-positive rate was 8.7% (2/23) in 2005, unexamined (?/6) in 2007, 0%(0/85) in 2009,and 0%(0/105) in 2010. Disinfection of contaminated animal rooms with formalin fumigation and Expor (Hypochlorous acid-based disinfectan) will be discussed about the eradication for Bacillus piliformis and those spores.

S 156 3P050-S Evaluation of Immunechromatographic Strip for Diagnosis of Helicobacter hepaticus Infection in Mice 1 1 1 2 ○Kanako Kato ,Takuro Aoki ,Akihiro Fuke ,Nobuhito Hayashimoto ,Fumihiro Sugiyama1,Ken-ichi Yagami1,Satoshi Kunita1,3 1Lab. Anim. Res. Cent., Univ. of Tsukuba, Tsukuba, Japan 2Central Inst. for Exp. Anim., Kawasaki, Japan 3Cent. for Exp. Med., Jichi Medical Univ., Shimotsuke, Japan

Aim: Helicobacter hepaticus (Hh) cause chronic hepatitis, hepatic tumor and inflammatory bowel disease in mice. A simple and rapid diagnostics is needed for in-house microbiological monitoring in laboratory mice, although PCR has been often applied. We evaluated specificity and sensitivity of immunochromatography(IC) strip to develop IC system as new diagnostics for Hh infection in laboratory mice. Materials & methods: IC strip was prepared with latex-conjugated anti-Hh monoclonal antibody and anti-Hh polyclonal antibody by fixing on nitrocellulose membranes. Pre-treated specimens of mouse feces were dropped into sample pad, and then result was judged visually after 10 min. Results & discussion: The IC strip specifically reacted to Hh, but not to Helicobacter bilis (Hb) and Helicobacter muridarum (Hm). It could detect at least 3500 cfu of Hh. Moreover, a comparative evaluation was carried out between IC and PCR using feces derived from experimentally-infected or naturally-infected mice. In the experimentally-infected mice, positive rate was 41/50 (82 %) in IC and 44/50 (88 %) in PCR, and diagnostic concordance rate between two methods was 39/50 (78 %). A sensitivity of IC compared to PCR was 19/39 (48.7 %) in naturally-infected mice. A specificity of IC was 43/54 (79.6 %).

3P051-S Identification of P.pneumotropica using by MALDI TOF-MS

○Hanako Morita,Nobuhito Hayashimoto

ICLAS Monitoring Center, Central Institute for Experimental Animals, Kawasaki, Japan

Purpose: In this study, we evaluated MALDI-TOF MS for identification of P.pneumotropica (Pp) because it is frequently isolated from the experimental animals. Materials and Methods: The 59 isolates of Pp (mice:44, Rats:15) was identified by default database (DB) in the MALDI Biotyper (Bruker Daltonics). The reference strains (ATCC35149, CNP160), and the 12 field isolates were used to set the new DB. And the 59 isolates of Pp were identified by default DB and new established DB. Furthermore, Other species in Pasteurellaceae that have possibility to be isolated from rodents were identified by the same way. Results and Conclusions: As a results, the average score of the 59 isolates by default DB were 2.023 (++), more than half isolates showed 'Genus Consistency' or 'No Consistency'. On the other hand, a result of adding new DB, the average score of the 59 isolates were 2.497(+++), most of the 59 isolates of Pp showed 'Species Consistency'. All strains of other species in Pasteurellaceae tested were not identified as Pp. From these results, application of the bacterial identification device, MALDI Biotyper, is useful for easy and rapid test for Pp in laboratory animals.

S 157 3P052-T Consideration of microbial contamination of sentinel animals in microbial monitoring

○Hikari Minamisawa, Hironori Izumi, Mayumi Adachi, Noriko Tsuneda, Hiroshi Yamamoto Division for Animal Resources and Development, Life Science Research Center, University of Toyama

In recent years, inter-facility transfer and breeding of genetically modified animals are increasing. To consider proper management of facilities for immunodeficient animals, we monitored sentinel mice for infection with Pseudomonas aeruginosa (P. A.) and Staphylococcus aureus (S. A.). Because ICR strains (from two breeding companies) were found to be contaminated with S. A., monitoring animals were replaced by a heterozygous nude strain in some experiments from 2013.

3P053-T Environmental monitoring: consideration on examinations of adhesion bacteria

○Chiemi Haneda, Shizuko Nagao

Education and Research Center of Animal Models for Human Diseases, Fujita Health University, Toyoake, Japan

In facilities where the laboratory animals are bred, it is necessary to be maintained with the proper management, so that the function of the institution is demonstrated. It should be the first prerequisite that a physical, chemical, environmental conditions are to be kept safe for the animals and breeders. It is reasonable that a microbiological control in the facilities bears the key role. For that purpose, environmental monitoring is important. In our institution, the environmental monitoring is carried out twice per year. In the monitoring, we are conducting adherent-bacteria measurement of doorknob etc., as breeding environmental agents. This movement of the bacteria to institutions should be prevented. The results of the monitoring is used as the quality assessment of institutions, or an index of periodical environmental monitoring. Various microbes are in our body being called as "resident bacteria". On the other hand, what lives in a temporary or special situation are called "transient bacteria". Generally, pathogenicity of "resident bacteria" is low, and "transient bacteria" adhere to fingers temporarily and become the source of infection easily. We are counting colonies of adherent bacteria is conducting in this Center. However, we believe that the adherent bacteria should be examined by investigating the strain of bacteria which bred. This action leads also to defense of movement of bacteria. So, we are examining adherent bacteria in this Center.

S 158 3P054-T Surveillance of Murine astrovirus infection in the Institute of Experimental Animal Sciences, Osaka University ○Yuko Kotani, Rieko Ando, Katsumi Aihara, Goro Hiraiwa, Yoko Mizuno, Shiro Kaneko, Soichiro Kagiyama, Akira Okamoto, Masaru Tajima The Institute of Experimental Animal Sciences Osaka University Medical School, Osaka, Japan

Murine astrovirus (AstV) was discovered in 1985. Outbreaks of AstV have not been reported in mice but in other animals with gastroenteritis and diarrhea symptoms. Thus, in this study we assessed the presence of AstV in mice from our SPF facilities and external facilities except for bleeding companies.

3P055-T Examination on the effect of intestinal protozoa extermination by metronidazole in mice (primary report) 1) 2) 1) 1) ○Yoshihiro Inoue , Akiko Tobinai , Takashi Ishibashi , Yohei Kudo , Yasuhisa Matsui 1) 1Institute for Experimental Animals (Division of Tumor Animals), 2 Department of Experimental Immunology, Institute of Development, Aging and Cancer (IDAC), Tohoku University [RATIONALE] Intestinal protozoa of mice have no pathogenicity to healthy mice. However, the management to prevent spreading of these protozoa between mouse breeding rooms in an institute is important, because some symptoms may occur in the immunodeficient mice by protozoan infection. In this study, we examined the extermination effect of metronidazole, which is an anti-protozoa drug for intestinal protozoa infected-mice, by feeding mice with solid diet containing metronidazole as a additive or with metronidazole-dissolved acid water. [METHODS] To investigate the extermination effect of metronidazole, protozoa-infected recipient mice (ICR) were generated. A cecal feces of protozoa-positive breeding mice were homogenated in saline, and the fecal solution (0.1 ml/mouse) was administrated four times, once a week, in oral into protozoa-free mice of 4-8 wk of age. Three experimental groups were set, solid diet with 0.1% metronidazole feeding group, acid water with 0.025% metronidazole feeding group, and untreated group (control group). After continuous five-week treatment, extermination of protozoa was analyzed. [RESULTS AND CONCLUSIONS] We were able to generate protozoa-infected recipient mice by the probability of 100%. However, all mice in metronidazole-treated group and control group had remained possessing of the intestinal protozoa. In brief, metronidazole treatment on these conditions were not effective to intestinal protozoa-infected mice.

S 159 3P056-S Analysis of Immunity in Common Marmoset Placenta

1 1 1 1 2 ○Shuya Mori ,Erina Numao ,Shino Ohshima ,Shin Shimada ,Junnko Okahara ,Erika Sasaki2,Ryuji Suzuki3,Hitoshi Ishimoto4,Takashi Shiina1,Yoshie Kametani1 1Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine,Tokai University School of Medicine, Kanagawa, Japan 2Central Institute for Experimental Animals, kanagawa, Japan 3 Sagamihara National Hospital, National Hospital Organization, kanagawa, Japan 4Department of Gynecology, Division of Specialized Medical Treatment, Tokai University School of Medicine, Kanagawa, Japan

In the pregnancy, mothers accept allogenic placenta dependent on a unique mechanism called immune tolerance in pregnancy. Because the placenta is evolutionally diverged and the HLA-G ortholog, a kind of suppressive MHC molecule, is not found in the rodents, the study including the molecules need to use non-human primates as model animals. We used common marmoset, a new world monkey, to analyze the immune system of primate pregnancy. We analyzed the cellularity of placental blood, mother blood and cord blood mononuclear cells by FACS analysis and compared with human ones. As a result, We found that the T cells are superior to B cells, and the ratio of CD4 T cells and CD8 T cells were nearly equal. The expression of CD3 in CD4 T cells had a bias to be lower than peripheral B cells. Classical class-I MHC expression was decreased in the fetus tissues compared with mother's decidua tissues. These phenomena are similar to human placenta's characters, suggesting that common marmoset is a good model animal for the analysis of MHC-dependent immune-tolerance in the pregnancy.

3P057-S Antibody production by transitional B cells developed in humanized NOG mouse

1 1 2 3 1 ○Mika Kojima ,Syuya Mori ,shino Ohshima ,Mamoru Ito ,Kiyoshi Ando ,Yoshie Kametani2 1Department of Hematology,School of medicine,University of Tokai,Kamagawa,Japan 2Department of Immunology,Division of Basic Medical Science and Molecular Medicine,University of Tokai,Kanagawa,Japan 3Experimental Animal Laboratoly,Kanagawa,Japan

Humanized mouse is a powerful tool for the analysis of human immune system in vivo. We have been investigated the functional development of B cells developed in NOG mice transplanted with human umbilical cord blood CD34+ cells (CB-NOG). We found that the B cells could not express mature B2 phenotype although the humanized mice serum contains natural IgM and IgG antibodies. In this study, we analyzed in what stage the B cells secrete natural antibodies. CB-NOG mice spleen cells were subdivided into the immature and transitional B cell fractions. These cells were stimulated with CpG and anti-IgM for 7 days and the IgM and IgG levels in the supernatants were examined. As a result, the immature and transitional B cells secreted IgM and IgG in vitro without acquiring the completely mature phenotype (CD21lo). The B cell fractions transplanted into NOG mice also secreted IgM and IgG without other human cell existence. These results suggest that the IgM and IgG antibodies are secreted by transitional B cells.

S 160 3P058-S Comparative analysis of hematopoiesis in c-kit-expressing stem/progenitor cells.

○Shino Ohshima,Shin Shimada,Syuya Mori,Mika Kojima,Asuka Miyamoto, Takashi Shiina, Asako Ando,Yoshie Kametani Department of Molecular Life Science, Division of Basic Medical Science, school of Medicine, Univercity of Tokai, Kanagawa, Japan c-kit is a receptor tyrosine kinase of stem cell factor, which plays a pivotal role in the hematopoietic stem/progenitor cell activity. As c-kit is known to have different expression profile between mouse and human hematopoietic stages, the molecule may have some species specific function. Thus, we compared the differentiation ability of human, common marmoset and swine c-kit-expressing cells. We employed two methods, one in vitro colony assay and the other in vivo xenotransplantation into immunodeficient NOG mice. As a result, all the cord bloods and bone marrow cells of the species contained c-kit+ cells, which possessed differentiation ability of erythroid and myeloid lineages in vitro. Moreover, they developed into the lymphoid lineage in the in vivo xenotransplantation. The bias to develop into erythroid lineage was observed in swine c-kit+ cells, while primate c-kit+ cells tended to develop into myeloid lineage in vitro. Human c-kit cells preferred to develop into B cells compared to T cells in vivo, while other species did not. These results suggest that the lineage bias of c-kit+ cells has species specificity.

3P059-S Association of the delayed humoral immune responses with the development of chronic GBV-B infection 1 1 2 3 ○Saori Suzuki ,Atsunori Higashino ,Ken-ichi Mori ,Yuko Katakai ,Akatsuki Saito1,Noboru Maki2,Hirofumi Akari1 1Primate Research Institute, Kyoto University, Inuyama, Japan 2Advanced Life Science Institute, Wako, Japan 3Corporation for Production and Research of Laboratory Primates, Tsukuba, Japan

It has been shown that humoral immunity, especially neutralizing antibody (NAb) response, is induced in the HCV infection and the NAb response may be associated with the control of the viral persistence. However, the precise kinetics in the induction of NAb is not fully understood. In this point of view, the animal model of HCV infection would be the key to solve this problem. GBV-B is closely related to HCV and GBV-B-infected primate model may bring important insight to clarify the implication of host immunity against Hepacivirus. In this study, we sought to examine the kinetics of humoral immune response in the course of acute and chronic GBV-B infection in marmosets and tamarins. We found that anti-core, E2 and NS3 antibodies were induced in the GBV-B-infected monkeys with sub-acute clearance, which crossed over the decrease of plasma viral RNA. In contrast, the antibody response in the persistently infected monkeys was much delayed. These results suggest that the inefficient induction of antibodies may be involved in the persistence of GBV-B infection. We assume that GBV-B may have an ability to retard humoral immune response by unknown mechanism aside from NAb escape.

S 161 3P060-S TLR9 ligand stimulation causes different responses in C57BL/6J and C3H/HeJ mice 1 1 1 2 2 ○Reiko Seki ,Kazuo Goto ,Teruko Fukuda ,Tamiko Yanagida ,Hajime Kono

1Depertment of Clinical Laboratory Science, Teikyo University Faculty of Medical Technology, Tokyo, Japan 2Depertment of Internal Medicine, Teikyo University School of Medicine, Tokyo, Japan

Objective; Toll-like receptors(TLRs) are essential for the development of innate and adaptive immune responses, and at least 13 TLR families exist in mice. However, the exact role of the TLRs remain unclear. C3H/HeJ have a misssense mutation in the Tlr4 gene, that results in a defective response to LPS stimulation. In this study, we investigated whether altered responses to TLR9 stimulation by CpG-DNA in C3H/HeJ protect them against liver damage in mice. Methods; C3H/HeJ(n=20) and C57BL/6J(n=36) at 6-10 weeks were injected intraperitoneally with 20ug CpG-DNA and 10mg or 20mg D-GalN. Blood samples for cytokine analysis were collected 1h after the injection. After 10h, the mice were killed, and livers sections were analyzed by H&E staining. ALT levels were measured using serum samples. Results and discussion; The survival rates at 10h after injection were 100% and 70.0% in the C3H/HeJ and C57BL/6J, respectively. Serum ALT levels in C3H/HeJ were significantly lower than those in C57BL/6J(P<0.01), and cytokine levels in C3H/HeJ were significantly lower than those in C57BL/6J (P<0.01;IL-1b,IL-10,GM-CSF,IFN-g,MIP-1a,MIP-1b, and TNFa, P<0.05;IL-2,IL-6, and IL-12p70). H&E staining results also showed reduced liver damage in C3H/HeJ. Conclusion; In this study, we found that C3H/HeJ mice have a lower response to TLR9 stimulation than do C57BL/6J mice.

3P061-S Characterization of human Eosinophil in HSC-transferred NOG-human IL-5 transgenic mice 1,2 1 1 1 ○Ikumi Katano ,Takeshi Takahashi ,Ryoji Ito ,Tsutomu Kamisako ,Yuyo Ka1,Tomoyuki Ogura1,Riichi Takahashi1,Hiroshi Suemizu1,Yutaka Kawakami2,Mamoru Ito1 1Central Institute for Experimental Animals, Kawasaki, Japan 2Keio University, School of medicine, Tokyo, Japan

We newly generated NOD/Shi-scid, IL2rgnull (NOG) mouse expressing the human interleukin-5 (hIL-5) gene, which is a differentiation and activation factor for eosinophil. We investigated human eosinophil development in this NOG-hIL-5 transgenic (hIL-5 Tg-NOG) strain after xeno-transplantation of human hematopoietic stem cells (HSC).NOG-hIL-5 Tg mice were generated by injecting the pCMV/hIL-5 DNA segment into prenuclear stage embryos of NOD-scid/+ IL2rgnull/+ mice, and further backcross-mated with NOG mice. Adult hIL-5 Tg-NOG and NOG mice were irradiated with 2.5Gy and then intravenously inoculated with 3.8-5 x 104 cord-blood derived human CD34+ progenitor cells. Human immune cells in peripheral blood (PB) of these mice were analyzed using flow cytometry. Human CD45+CD66b+CD16-CD125w(IL-5Rα)+ eosinophil was detected in PB of HSC-transferred hIL-5 Tg-NOG mice at 4 weeks, but not in NOG mice. Human eosinophil constituted 7.8±4.0% of the total mononuclear leukocytes of hIL-5 Tg-NOG mice at 12 weeks after HSC transfer, whereas less than 0.5% in NOG mice. Giemza staining confirmed that human eosinophil had bilobed nuclei and contained many red-stained cytoplasmic secretory granules in PB of HSC-transferred mice.These data suggest that NOG-hIL-5 Tg mice might become a useful tool for analysis of human eosinophil in vivo.

S 162 3P062-S The effects of mast cell on environmental particulate-induced respiratory allergy

○Risako Nishino,Tomoki Fukuyama,Yuko Watanabe,Yoshimi Kurosawa, Hideo Ueda,Tadashi Kosaka,Takanori Harada Institute of Environmental Toxicology, Ibaraki 303-0043, Japan

The inhalation of many types of environmental particulate is a cause of respiratory allergy and the protocols are needed. Previously, we developed a new detecting method using long term dermal sensitization with an intratracheal challenge. To improve our method, we compared the allergic effects between Balb/c and NC/Nga mice using our method and pulverized chemical respiratory allergen, trimellitic anhydride (TMA). Mice were sensitized dermally followed by inhaled challenge with TMA. Animals were carefully observed during inhaled challenge. On the day after last challenge, all mice were sacrificed and measured IgE levels, immunocyte counts and cytokine/chemokine levels in serum, hilar lymph nodes and bronchoalveolar lavage fluids. As the results, the TMA-sensitization/challenge groups of both strains were significantly increased in all endpoints compared to each control group and these results show that our method can be used to detect allergic responses to inhalation of environmental particulate. However, worsening of respiratory statuses with marked increase in mast cells and related factors were noted only in NC/Nga mice. In conclusion, it suggested that the results of sensitization assays with environmental particulate might be greatly influenced by the animal strains used and mast cells might be deeply involved in the aggravation of airway allergic symptoms induced by environmental particulate.

3P063-S Quantitative Analysis on Immunological Profile in NOG-hairless Mice 1 2,3 2 2,3 ○Kazuhiko machida ,Shinji Kusakawa ,Rumi Sawada ,Satoshi Yasuda ,Youji Satou2,3,4,Hideki Tsutsumi1 1Central Institute for Experimental Animals 2National Institute of Health Sciences, Tokyo, Japan 3Foundation for Bionedical Research and Innovation, Kobe, Japan 4Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan

We conducted a quantitative analysis on immunological profile in NOG-hairless (NOD/Shi-scid IL2Rgnull-hr, hereinafter NOG-hr) mice and compared with the data in NOG mice that we had reported at the last meeting. Eighty male NOG-hr mice were assigned to 4 and 4 groups, HeLa group (The number of the inoculated cells: 0, 103, 104, 105/mouse) and HeLa + Matrigel (MG) group (0, 101, 102, 103/mouse), respectively. Each mouse was inoculated with each prescribed cells subcutaneously and observed nodule formation for sixteen weeks. The TPD50 (tumor-producing dose at the 50% endpoint) was analyzed by 4-parameter logistics. The results show that TPD50 of the HeLa cells or HeLa cells mixed with MG in NOG-hr mice was 1/13 (3.2×104/4.2×105) or 1/2040 (2.1×102/4.2×105), respectively. This suggests that the level of immunological deficiency in NOG-hr mice was lower than that in NOG mice, 1/33 (1.3×104/4.2×105) or 1/5431 (7.8×101/4.2×105), respectively. We also compared the differentiating ability of human CD34+ hematopoietic stem cells from umbilical cord blood in these two strains, and obtained the result which supported mentioning above.

S 163 3P064-S Morphological studies of the nasal cavity and olfactory epithelium of the Zebra finch

○Makoto Yokosuka,Tomoaki Nakada,Kyohei Mikami

Department of Comparative Medicine, Nippon Veterinary and Life Science University, Tokyo, Japan

Because the zebra finch (Taeniopygia guttata) is commercially available, easy to breed in captivity and robust to anaesthesia, this bird has become the measure model for many neuroscience studies especially for human speech and learning. However, until now, only a few studies have investigated the chemical sensory apparatus of this bird, so little is known of the types of chemical senses that it uses. Thus, we analyzed the anatomical and histological properties of the nasal cavity and olfactory epithelium of the zebra finch to investigate its functional level of the olfaction. In the nasal cavity of the zebra finch, although the anterior and maxillary conchae were clearly observed, there was obscure structure equivalent to the posterior concha. The olfactory epithelium of the zebra finch occupied remarkably small area of the posterior concha and had the histological and ultrastructural features of the olfactory receptor cells like already reported other avian species. These morphological and histochemical properties were reflected like the bird of the passeriformes, such as the Japanese and/or American crows and the brown-eared bulbul. Our results suggested that the zebra finch has a limited sense of olfaction, as same as other passeriforme avians. Moreover, zebra finch's olfactory system may offer a unique model for studying the evolution and development of the vertebrate olfactory system.

3P065-S Analysis of traumatic focal brain injury in Cav2.1

channel α1 subunit mutant mice 1 1 2 ○Tae Yeon Kim ,Eiki Takahashi ,Chitoshi Itakura

1Support Unit for Animal Resources Development, Brain Science Institute, RIKEN, Japan 2Research Resources Center, BSI, RIKEN, Japan

2+ Abstract Voltage-gated Ca (Cav) channel is a molecular complex consisting of α1, α2δ, β, and γ subunits. It plays an important role in the regulation of diverse neuronal functions which are related with intracellular Ca2+ concentrations and also in glutamatergic synaptic transmission. In ischemic brain damage, glutamate triggered excitotoxicity plays an important role. N-methyl-D-aspartate (NMDA) receptor has been considered as the key player in this process and in neuronal death. In a research using ischemic model of rolling Nagoya mice, one of Cav2.1α1 mutant mice, showed decreased neuronal death compared to wild type mice. Here we used Tottering-j6 mice which are chemically-induced mutant strain produced using ethylnitrosourea (ENU). We made in vivo focal traumatic brain damage model by using a cryogenic method. Due to an early damage of the blood brain barrier (BBB), the cryogenic cortical injury leads to vasogenic edema, marked brain swelling, and inflammation. We injected Evans Blue to assess the BBB damage. The region stained with Evans Blue was smaller in Tottering-j6 mice than wild type mice. In addition, the signal of Fluoro Jade B, the marker of neuronal damage, and anti-glial fibriallary acidic protein (GFAP), the marker of reactive astrocytes were weaker. These results demonstrate that the mutant Cav2.1 channel in Tottering-j6 mice plays a protective role in a Ca2+- dependent manner.

S 164 3P066-S Behavioral genetics in Japanese wild mouse-derived strain MSM/Ms reveals the evolution of PACAP gene 1,2 1,2 1,2 1,2 ○Akira Tanave ,Ayako Ishii ,Aki Takahashi ,Tsuyoshi Koide

1Mouse Genomics Resource Laboratory, National Institute of Genetics (NIG), Shizuoka , JAPAN 2Department of Genetics, School of Life Science, The Graduate University for Advanced Studies, Shizuoka, Japan

We previously conducted open-field behavioral tests, using consomic strains of mouse, each of which has replaced single chromosome of MSM on C57BL/6 (B6) genetic background. We found that B6-Chr17MSM consomic strain, which carries MSM-derived Chr17, shows high anxiety-like behaviors.We developed a series of sub-consomic strains, which carries a partial region of Chr17 derived from MSM. Open-field behavioral tests successfully mapped a genetic locus into about 2Mb region in Chr17, in which only one protein coding gene, PACAP, are located. Quantitative real-time PCR analysis and radio immunoassay showed significantly increased PACAP expression levels in hypothalamus of sub-consomic mice. We found a microsatellite mutation in MSM strain as a cause of the increased expression levels. Corticosterone levels associated with the PACAP expression were significantly enhanced after restrained stress in sub-consomic mice. The lengths of microsatellite repeat were clearly shorter and the genetic diversity in PACAP gene was significantly decreased in laboratory than wild-derived strains.These results suggesting that behavioral selection during the domestication of mice resulted in the change of PACAP gene diversity associated with the decreased stress response.

3P067-S Live-imaging analyses of SIL1-deficient neuron migration

○Yutaka Inaguma,Nanako Hamada,Hidenori Tabata,Hidenori Ito, Makoto Mizuno, Koh-ichi Nagata Institute for Developmental Research, Aichi Human Service Center, Kasugai, Japan

Mental retardation (MR) affects about 1-3% of the general population. Even if causative genes of MR are identified, lack of information of underlying molecular mechanisms impedes the development of new treatments and diagnosis methods. SIL1 gene, encoding a nucleotide exchange factor, regulates a chaperone HSPA5 as a co-chaperone. Mutations in the SIL1 were identified as a major cause of Marinesco-Sjogren syndrome (MSS). MR is a characteristic symptom of MSS. Since abnormal cytoarchitecture is observed in the cerebral cortex of MSS, we performed RNAi experiments to examine the role of SIL1 in the migration of newly-generated cortical neurons. RNAi vectors and pCAG-EGFP were coelectroporated into progenitor cells in ventricular zone of embryonic murine brains by the in utero electroporation method, and localization of transfected cells and their progeny was observed at P0 and P7. Consequently, we revealed SIL1-silencing caused neuronal migration delay during corticogenesis. While RNAi-resistant SIL1 rescued the defects, three MSS-causing SIL1 mutants tested did not. When SIL1 was silenced in cortical neurons in one hemisphere, axonal growth in the contralateral hemisphere was delayed at P7. Furthermore, time-lapse imaging analyses ex vivo revealed morphological disorganization associated with abnormal migration of SIL1-deficient neurons. Taken together, abnormal neuronal migration and interhemispheric axon development may contribute to MR in MSS.

S 165 3P068-S Suppression of dopamine D1 receptor expression causes decreased motor activity in mice 1 2 2 2 ○Toshikuni Sasaoka ,Asako Sato ,Satoshi Arai ,Jun Maeshima ,Tadashi Okubo2,Nobuyoshi Fujisawa1,Toshiya Sato1,Kanako Oda1,Yoshitaka Maeda1,Minoru Tanaka1,Yoshitaka Yamamoto1,Seiko Sakai1,Yukihiro Jinbo1,Saori Chiba1,Minesuke Yokoyama1,3 1Dept Comp & Exp Med, Brain Res Inst, Niigata Univ, Niigata, Japan 2Dept Lab Anim Sci, Kitasato Univ Sch Med, Sagamihara, Japan 3Cent Inst Exp Anim

Dopamine controls a wide variety of behavior related to motor control. Dopamine D1 (D1R) and D2 receptors (D2R) are found at high levels in the striatum, which have important roles in motor control. Although D1R signal theoretically promotes motor activity, D1R knockout (KO) mice are hyperactive. It is assumed that potential developmental adaptation could cause hyperactive phenotype in D1R KO mice. To circumvent developmental adaptation, we generated a transgenic mice harboring tetracycline-regulated expression of D1R gene with D1R KO background (D1R -/-;Tg +). Without doxycycline (Dox) the transgenic mice showed high expression of D1R, which rescued hyperactive phenotype in D1R KO mice. To clarify the role of D1R in adult mice, we suppressed the D1R expression in adult D1R -/-;Tg + mice, and home cage activity was examined. When Dox was administered, D1R expression was decreased to levels undetectable. Simultaneously, home cage activity level was significantly decreased. These results demonstrate that D1R-mediated signal is required to maintain normal motor activity. The D1R -/-;Tg + mice will be a useful animal to investigate the role of D1R in adult mice with less influence of developmental adaptation.

3P069-S Behavior Observation of Pygmy Marmoset under the Laboratory Environment

○Yukiko Abe, Atsu Aiba, Kazuki Nakao

Section of Animal Research, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, the University of Tokyo, Tokyo, Japan

Pygmy marmoset is a one of the world's smallest primates and mainly bred in zoos in Japan. Although it has been reported of breeding them in laboratory animal facilities in other countries, no report can be found in Japan. Thus we are trying to breed pygmy marmoset under the laboratory environment. As the first step, we observed the behavior of pygmy marmoset under laboratory environment. Pygmy marmosets, aged 18months to 60months, accommodated to the 12h light : 12h dark cycle immediately after they put in the laboratory environment, that is about 15min after the beginning of dark phase they quitted the activities and stayed as they were during dark phase, and started activity about 15min before the beginning of light phase. Eating behavior was only seen during light phase. Females spent outside the nest longer than male and frequently accessed to the feedboxes. We will also report their behavior changes when the light/dark cycle was changed from 12h light: 12h dark to 10h light: 14h dark or 8h light: 16h light.

S 166 3P070-S Histological analyses of age-related ovarian pathologies in SOD1-deficient female mice 1 2 2 2 ○Yusuke Ozawa ,Ikko Kawashima ,Naoki Okamoto ,Kazuhiro Kawamura ,Takahiko Shimizu1 1Department of Advanced Aging Medicine, Chiba University Graduate School of Medicine, Chiba, Japan 2Department of Obstetrics and Gynecology St. Marianna University School of Medicine, Kawasaki, Kanagawa

Superoxide dismutase 1 (SOD1) is one of the cellular antioxidant enzymes, and is distributed in the cytoplasm to eliminate superoxide. We previously reported that Sod1-/- female mice showed abnormal luteal formation and decreased plasma progesterone level resulted in infertility. In the present study, we histologically investigated age-related ovarian changes in Sod1-/- female mice. Although Sod1-/- females showed no morphological abnormalities alities in diapause ovary at 3 months of age, they exhibited a significant reduction in ovarian weight at 6 to 10 months of age. Histological analyses revealed that no difference in number of primordial, primary, secondary, and antral follicles was observed between Sod1-/- and Sod1+/+ female mice. Interestingly, Sod1 deficiency selectively decreased number of the corpus luteum in ovary. In addition, we induced hyperovulation to count oocyte number in Sod1-/- females by gonadotropin injection at 6 months of age. We detected no difference in oocyte number between Sod1-/- and Sod1+/+ females. These results suggested that cellular oxidative stress in ovary by Sod1 loss impaired luteal formation leading to ovarian atrophy during aging in Sod1-/- female mice.

3P071-S Aged animals fostering in the new breeding environment 1 1,2 1,2 1,2 ○Yoshihiro Noda ,Sinji Tokima ,Daijyu Mutou ,Yui Miyazawa ,Naoko Maekawa1,2,Yoko Takahashi1,2,Kazunao Kuramoto1,Tamao Endo1 1Animal Facility, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan 2KAC CO., LTD., Tokyo, Japan

Aged animals breeding are important in gerontological research. As for fostering of an aged animal breeding throughout the life from the juvenile age period, long-term stable environment is required, but temperature-humidity management and the microbe control are not easy by facilities administration. In Tokyo Metropolitan Institute of Gerontology, since 1979, mice (C57BL/6NCrSlc, B6D2F1/Crlj, C57BL/6J), and rats (Wistar, F344/DuCrlCrlj) have been brought up into aged animals. Average lifespan data of the B6D2F1 were 870 days (male), 783 days (female) and those of C57BL/6NCr 826 days (male), 766 days (female). In rats, average lifespan of the Wistar were 810 days (male), 927 days (female) and those of Fischer were 859 days (male), 946 days (female).New facilities opened our research institute in June, 2013, and, in the built laboratory animal facilities, an individual ventilation cage (IVC) system was introduced as standard breeding equipment. The breeding environment changes the cage from aluminum to the plastic. The feed is changed to Low Irradiated Diet by autoclave sterilization. Animal bedding is changed to the paper tip by a wooden chip. The water to drink was changed from bottle of the chlorine hydrochloric acid addition water to chlorine addition Automated Watering System. In this study, it was further investigated whether the change of the breeding environment influenced Aged animals breeding.

S 167 3P072-S Mouse Hepatitis Virus Infection in a Laboratory Animal Facility

1 1,2 1,2 1,2 ○Yuka Ishida ,Takashi Okubo ,Shun Kawahara ,Kaori Tateno ,Tatsuo Hayao1,Wataru Ueno1,Seiji Kito1,Toshiaki Kokubo1 1Laboratory Animal and Genome Sciences Section, Dept. of Technical Support and Development Research, Development and Support Center, National Institute of Radiological Sciences, Chiba, Japan 2Science Service Inc., Tokyo, Japan

Animal Research Building of semi-barrier system is one of the laboratory animal facilities in National Institute of Radiological Sciences. The frequency of microbiological monitoring tests in this facility is quarterly. We were recognized Mouse hepatitis virus (MHV) infection in four rooms among 12 mouse breeding rooms in October, 2013. We carry out cleaning of this facility and preventing the spread of infection as follows. I. The cleaning of this facility carries out each four breeding areas. II. The mice that were not able to sacrifice immediately put it into two infection breeding rooms or three non- infection breeding rooms. Then, the mice are sacrificed within a fixed period of time. We report on a current detailed and present correspondence.

3P073-S Bedding coated with propolis inhibits intracage bacterial growth and occurrence of mouse allergen 1 2 3 ○Noriko Tosa ,Kumiko Yoshimatsu ,Tadasuke Tsukiyama ,Shigetsugu Hatakeyama3,Jiro Arikawa1,3 1Institute for Animal Experimentation, Hokkaido University Graduate School of Medicine, Sapporo, Japan 2Department of Microbiology, Hokkaido University Graduate School of Medicine 3Department of Biochemistry, Hokkaido University Graduate School of Medicine

Laboratory animal allergy (LAA) is a form of occupational allergic disease. Several studies indicate that another substance, lipopolysaccharide (LPS), a cell-wall product from Gram-negative bacteria, is related to promote sensitization to the allergens. Propolis is a natural resinous mixture produced by honey bees and its antibacterial activity has been well known. In this study, we investigated whether bedding coated with propolis inhibits the bacterial growth and the occurrence of allergens inside cages. Intracage temperature, humidity, particle number, and ammonia level were no difference between the cages including bedding coated with propolis and that including non-coating bedding. On the other hand, bacterial number and mouse allergens (albumin) decreased in the cages including bedding coated with propolis compared with that including non-coating bedding (P=0.0265, P=0.0015, respectively). Moreover, mouse allergens decreased compared with before coating bedding with propolis (P<0.0001). In summary, the bacterial growth and the occurrence of allergens inside cages were inhibited by coating bedding with propolis. The findings indicate the possibility that the bedding coated with propolis is useful for the preventive measure against LAA.

S 168 3P074-S Application of mouse sperm cryopreservation at CARD, Kumamoto University 1 1,2 1,2 1,2 ○Eri Kohagura ,Kiyoko Fukumoto ,Yukie Haruguchi ,Tomoko Kondo ,Yumi Takeshita1,2,Yuko Nakamuta1,2,Tomoko Umeno1,2,Mari Iwamoto1,Fumi Takahashi1,Syuuji Tsuchiyama1,Toru Takeo1,Naomi Nakagata1 1Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto, Japan 2Kyudo Co., Ltd., Tosu, Japan.

Our center has provided a mouse bank service to facilitate scientific research using genetically engineered mice (GEM) for researchers. At our center, we have archived over 3,000 strains of embryos and sperm of GEM since we started the mouse bank system in 2000. Recently, sperm cryopreservation is preferably selected to preserve GEM. There are many reasons for choosing sperm cryopreservation, such as the ability to store many sperm collected from one male, the ease of carrying out the freezing and thawing procedures, and the ability to produce many fertilized eggs via IVF. In addition, we have developed highly efficient sperm cryopreservation and IVF techniques to obtain high fertilization rates. In this presentation, we will introduce our mouse bank system and provide useful information for the effective use of our service.

3P075-S Examination of a Method of Strain Storage Useing Frozen Sperm of BALB/cByJ Mice

○Mutsumi Yamane,Kazuhiro Kaseda,Ayako Isotani,Masahito Ikawa

Osaka University

Recently, the CRISPR/Cas method was developed as a technique for developmental engineering, and it enables us to create genetically engineered animals in a simple and timely manner. In the future, as explosive growth of the number of the animals generated is expected, simple and secure methods of strain storage will become necessary to stockpile them as bio-resource. Therefore, we focused on sperm cryopreservation which is an easy technique. However, sperm cryopreservation needs to undergo in vitro fertilization (IVF) to generate a living animal. Thus we examined the media conditions used in IVF by using frozen sperm of BALB/cByJ mice, a strain that was reported to have low fertility rates. We examined four media conditions of IVF. Frozen sperm of BALB/cByJ was thawed, incubated in preincubation media and transferred to the insemination media including oocytes. Fertilization rates were calculated by counting number of 2-cell embryos. Moreover, we also used CARD media with an increased supplement of 30µL. The embryos were transferred into pseudopregnant mice, and we confirmed whether offspring were obtained. The fertilization rates of combinations including mHTF media, which is a general-purpose media, was 32.7%. In contrast, the combinations including FERTIUP and CARD media (spp. 15µL) was 87.5%. In addition, healthy offspring were obtained by embryo transfer when CARD media (spp. 30µL) was used. From the above results, Sperm cryopreservation is a viable method of storing low fertility performance strain.

S 169 3P076-S RIKEN BRC JAPAN MOUSE CLINIC: Comprehensive Mouse Phenotyping for Research supports and for IMPC 1 1 1 1 ○Tomohiro Suzuki ,Hideki Kaneda ,Kimio Kobayashi ,Ikuo Miura ,Tamio Furuse1,Ikuko Yamada1,Osamu Minowa2,Hideaki Toki2,Nobuhiko Tanaka3,Masaru Tamura1,Hiroshi Masuya3,Shigeharu Wakana1,Yuichi Obata4 1RIKEN BRC, Technology and Developmental Team for Mouse Phenotype Analysis, Tsukuba, Japan 2RIKEN BRC, Team for Advanced Development and Evaluation of Human Disease Models, Tsukuba, Japan 3RIKEN BRC, Technology and Development unit for Knowledge Base of Mouse Phenotype, Tsukuba, Japan 4RIKEN BRC, Tsukuba, Japan

We have operated the Japan Mouse Clinic (JMC) aimed to evaluate comprehensive and detailed phenotypic characteristics for the mouse resources of the mouse research communities. A variety of phenotyping data are obtained in two phenotypic pipelines, one is fundamental / in depth pipeline and the other is behavior oriented pipeline. A lot of phenotypic data from approximately 140 strains contributed to findings in mouse research communities. International Mouse Phenotyping Consortium (IMPC) is an International project aimed to carry out high-throughput phenotyping of each line in order to determine the function of every gene in the mouse genome. The IMPC has also the international project for distributing the phenotypic data and mouse to researchers of the world. We have already started analyzing a several knock out mouse lines in IKMC resources. A vast amount of phenotyping data in the analyzed mouse lines have been released on JMC homepage using the web-based database named "Pheno-Pub". We also report the results of phenotyping analyses of various mouse resources in JMC.

3P077-S Development and analysis of the database of the Exchangeable Gene Trap Clones (EGTC)

○Masatake Araki,Mai Nakahara,Chika Yanai,Fumika Yamazoe, Mikiko Miyake, Ayaka Morita,Miyuki Araki,Yoshiyuki Okamoto,Naomi Nakagata, Kumiko Yoshinobu, Ken-ichi Yamamura,Kimi Araki IRDA, Kumamoto University, Kumamoto, Japan

Background and Purposes: We have established an exchangeable gene trap system, in which a reporter gene can be exchanged for any other DNA of interest. We constructed the database for the Exchangeable Gene Trap Clones (EGTC) [ http://egtc.jp ]. Because we used a promoter-trap strategy, all trapped genes were expressed in ES cells, and this restriction might affect the diversity of gene function among the trapped genes. Therefore, we examined the diversity of trapped genes in EGTC. Methods: We did KEGG pathway analysis and Gene Ontology (GO) analysis of the EGTC trap clones. Results: Among the 800 EGTC genes that mapped to KEGG genes, 289 genes were assigned to 932 KEGG pathways. EGTC trapped genes were found in almost all the pathways. There was no apparent gene function bias among the trapped genes. In all three ontologies, biological process, cellular component, and molecular function, the top two most frequent second-level categories were identical between the trapped genes in the EGTC mouse lines and RefSeq genes, indicating that there was no special bias in the trapped genes in EGTC lines. In addition, the low frequent categories under 1% were also identical between the trapped genes and RefSeq genes. Conclusions: The functional distribution of trapped genes in EGTC mouse lines have similar tendencies as the RefSeq genes for the whole mouse genome.

S 170 3P078-S Effects of imposed exercise on a blood lipid level and spontaneous activity in European wood mouse

○Mizuho Inokuchi,Hiroki Shichijo,Shinsuke Sakamoto,Akio Shinohara,Chihiro Koshimoto Frontier Science Research Center, University of Miyazaki, Miyazaki, Japan

Aim: Laboratory mouse and rat are less qualified for an animal model of human hyperlipidemia because of their specific lipid metabolizing system. We have been maintained European wood mouse (Apodemus sylvaticus) as a closed colony, some of which develop hyperlipidemia spontaneously. The hyperlipidemic animals (Apodemus hyperlipidemia: AHL) have lower activity mass than the animals with normal cholesterol level (Apodemus normolipidemia: ANL) in this species. In this study, we made treadmill exercise test to reveal a lipid metabolism characteristic in this species. Materials and methods: Male animals were used in this 4 weeks experiment (AHL: n=14, ANL: n=6). The first half is a control period and the second half is an exercise loading period. Daily food and water intakes as well as weekly body mass change were monitored. The sample blood was taken on day 0, 14 and 28 to assay blood lipid profile. Spontaneous activity was monitored throughout the experimental period. Results and discussion: In both periods, the spontaneous activity of AHL tend to be less than that of ANL (P=0.10,P=0.28). Spontaneous activities were significantly reduced in exercise period in both groups (P<0.05,respectively). The weights were significantly decreased in both groups, although total cholesterol levels were not significantly changed(P=0.29,P=0.22), indicating that hyperlipidemia in this species was not improved by the exercise.

3P079-S Resource development by high-throughput mutation detection: Use and comparison of several NGS 1,2 1 1 1 ○Hayato Kotaki ,Ryutaro Fukumura ,Takuya Murata ,Shigeru Makino ,Yuji Nakai1,Yuichi Ishitsuka1,Atsushi Toyoda2,Hideki Noguchi2,Asao Fujiyama2,Yoichi Gondo1 1RIKEN BRC 2National Institute of Genetics

RIKEN RGDMS (http://www.brcriken.jp/lab/mutants/jp/genedriven.htm) have been providing mutant mouse strains with base-substitutions on requested target genes. It consists of dual archives of frozen sperm and genomic DNA of ~10,000 ENU-mutagenized G1 mice. We screen mutations on the user's target gene then provide live heterozygous mutant mice by IVF/ET method based on the user's request. To accelerate this system, we have adopted the next-generation sequencers (NGS) and succeeded in comprehensively identifying ENU-induced mutations in G1 exomes. We have so far used HiSEQ2000, SOLiD4 and Ion Proton with the SureSelect Mouse Exome Target Enrichment System that covers 49.6 Mb of mouse coding sequences. Here, we report the comparison of the three systems with an identical G1 genomic DNA sample. The HiSEQ2000 (100 bp PE), SOLiD4 (50 bp SE) and Ion Proton (~120 bp SE) gave 200, 110, 40 millions reads with more than 10-fold reading coverage of 99.8%, 88.3% and 96.3% of the 49.6-Mb exome, respectively. The tested G1 has the DBA/2JXC57BL/6J genetic background; thus, the known dbSNPs between the two strains provide the positive control dataset. The detection rates of the dbSNPs were 97.3%, 86.0% and 93.9%, respectively. The detected ENU-induced mutations were 87 and 49 by HiSEQ and SOLiD, respectively. The detected number by Ion Proton is under analysis and will be presented together.

S 171 3P080-S Resource development by high-throughput mutation detection: discovery of spontaneous mutations ○Yuichi Ishitsuka,Shigeru Makino,Hayato Kotaki,Ryutaro Fukumura, Yuji Nakai, Yoichi Gondo Mutagenesis and Genomics Team RIKEN BioResource Center, Ibaraki, Japan

Various established strains, e.g., inbred strains, have tremendously contributed experimental animal sciences. Even in inbred strains, however, each animal accumulates spontaneous mutations every generation. At present, it is widely accepted that the spontaneous mutation rate in human and mouse is roughly 1X10-8/bp/generation. Each diploid zygote thus carries ~60 new mutations per generation in human and mouse. We have already established a next-generation sequencing (NGS) method to detect such de novo germline mutations focusing on 49.6-Mb mouse exome. We adopt this system to identify spontaneous mutations in C57BL/6J strain. The establishment of the spontaneous mutation discovery system should contribute the quality control of resources, the assessment for various environmental mutagens including low-dose radiations, and the development of novel disease models. We started from 16 independent pairs and have been keeping the mating so that the inbreeding coefficient becomes zero or minimum. After 5 generations without selective pressures on recessive mutations, we will detect the de novo mutations by NGS. All the mice in this mating scheme will be kept frozen so that it is possible to trace the origin of each detected mutation in the pedigree. It is expected for each mice of the 5-th generation to have 300 de novo mutations that occurred somewhere in this pedigree, if the mutation rate is truly 1X10-8/bp/generation.

3P081-S Immunological screening in International Mouse Phenotype Consortium (IMPC)

○Mao Ozaki,Tomoko Kagami,Eiji Oka,Osamu Minowa,Setsuko Mise, Takahiro Doi, Shigeharu Wakana RIKEN BioResource Center (BRC)

The IMPC has carried out high-throughput phenotyping of each line in order to determine the function of every gene in the mouse genome. The IMPC has also the international project for purpose of distributing the phenotypic data and mouse to researchers of the world. We show the immunological screening using multicolor flow cytometry.Spleen cells were harvested from 16 weeks mice and stained with 17 antibodies (2 panels, 8 color/each panel) for detection of the profiling data on T cells, B cells, NK cells, Dendritic cells and subpopulations of each cell type, total 29 subpopulations. The optimized conditions were set through the control experiments of compensation and fluorescence minus one to obtain the reproducible results. We analyzed C57BL/6N Tac (B6/NTac), a basal line in IMPC, substrains of B6 (JJcl, NJcl, JCrl, NCr) and as a outlier BALB/cAjcl. There were two clusters; one included all B6 strains and the other is BALB. There were several clusters among B6 substrains (males) up to the bleeders. The results of analysis for NOD/Shi-scid revealed that the relative weight and numbers of nucleated cells in spleens were significantly decreased compared to B6 strains.Our results show that it is a high-throughput analysis of immune cells with our multicolor flow cytometry and that is useful for clarification of the difference between normal and mutant mice. We are going to accomplish together with the other institutions for the best SOP and obtaining the data with higher accuracy.

S 172 3P082-S Laboratory Animal Resource Bank at NIBIO for drug discovery and human health

○Minako Koura,Akiko Kawai,Masafumi Nakano,Yoko Noguchi, Osamu Suzuki, Junichiro Matsuda Laboratory of Animal Models for Human Diseases, National Institute of Biomedical Innovation, Osaka, Japan

The National Institute of Biomedical Innovation (NIBIO) was established in 2005 by the Ministry of Health, Labour and Welfare, for the purpose to contribute to the creation of the innovative pharmaceuticals and the improvement of the national health. We established the Laboratory Animal Resource Bank at NIBIO in 2006 for promoting the drug discovery and human health. We have been conducting the collection, maintenance, preservation, supply and database construction of disease model animals, especially mice. At present, more than 200 mouse strains are deposited to our bank and their embryos/sperm are safely cryopreserved. In some spontaneous mutants, in vitro fertilization is difficult to apply. In such a case, we have to maintain the mice by natural mating and confirm the disease onset, and then we distribute the mice to researchers with disease and breeding information. We also provide the safe deposit service of mouse strains to researchers by cryopreserving sperm/embryos of mice on condition that their information is not open to the public. The cumulative number of access to our home page http://animal.nibio.go.jp/ reaches more than 500,000. We are also contributing databases which are available from http://mbrdb.nibio.go.jp/cgi-bin/index.cgi, http://www.shigen.nig.ac.jp/mouse/jmsr/, http://sagace.nibio.go.jp/.We will give an overview of our activities in the poster.

3P083-S Use of mouse strains preserved by sperm freezing

○Maiko Ijuin,Kyuichi Taguma,Michiko Hashimoto,Rika Takashima, Noriko Hiraiwa, Kazuyuki Mekada,Ayumi Hasegawa,Keiji Mochida,Atsuo Ogura,Atsushi Yoshiki RIKEN BRC

Genetically modified mice are efficiently preserved by sperm freezing. RIKEN BRC has promoted cryopreservation by sperm freezing for mouse strains of C57BL/6, DBA/2, C3H, BALB/c, FVB, and F1 backgrounds. We report here our protocols of sperm freezing and how to use frozen sperm distributed from RIKEN BRC. Sperm freezing Using the method of Takeshima et al (1991), epididymal sperm was suspended in an 18% raffinose and 3% skim milk solution, embedded in straws, and frozen. The sperm was measured for motility, concentration, and proportion of immotile sperm and classified into six ranks. In vitro fertilization with frozen-thawed sperm A straw containing the frozen sperm was immersed in a water bath at 37o for 15 minutes. Recovered sperm solution was transferred to a drop of HTF medium containing PVA and 0.4 mM methyl-β-cyclodextrin and incubated for 1 hour. Oocytes were collected and incubated in drops of mHTF-1.25mM reduced glutathione medium. Three hours after insemination, morphologically normal oocytes were transferred into CZB medium containing glucose. The fertilization rates of frozen-thawed sperm of C57BL/6J congenic strains were 62.8-82.7%. Use Frozen sperm or recovered litters from frozen sperm are available via the RIKEN BRC website. In order to distribute high-quality mouse resources by using frozen sperm, we will use frozen sperm of excellent motility and highest concentration. Please submit your request by e-mail to [email protected].

S 173 3P084-S Microbiological Status of Mouse Strains in RIKEN BRC and Rederivation in 2013

○Noriko Hiraiwa,Ayako Kajita,Naoki HIirano,Akemi Yasui, Yasutaka Noda, Tatsunori Yamamura,Kazuyuki Mekada,Fumio Ike,Atsushi Yoshiki Experimental Animal Division, RIKEN BioResource Center, Tsukuba, Japan

RIKEN BioResource Center (BRC) has started collection, preservation and distribution of high-quality mouse resources since 2002 and collected over 7,000 strains of mice until now. In our workflow, deposited mice from any microbiological environments are serologically tested (Mouse hepatitis virus (MHV), Mycoplasma pulmonis, Clostridium piliforme, Sendai virus, Ectromelia virus, Hanta virus, Lymphocytic choriomeningitis virus and Salmonella typhimurium), and then housed in the specialized conventional area aiming to prepare enough size of colonies for rederivation by embryo transfer (ET) or Caesarian section (CS) treatments. Parasitology (intestinal protozoa and pinworms) is carried out using cecum contents taken at the rederivation step. After six weeks nursing of pups obtained by ET and/or CS treatments in the barrier quarantine, foster mothers are tested to judge pups free of at least 18 agents specified by BRC. In 2013, we received 152 strains from SPF and 15 strains from conventional facilities. MHV and M. pulmonis were not detected among deposited mice. However, intestinal protozoa and pinworms were prevalently noted (118 strains) both in SPF and conventional derived mice. After rederivation, removal of all microbiological contaminations observed at the deposition was confirmed. We will keep clean microbiological status in BRC by brushing up our rederivation workflow from now on.

3P085-S Current status of RIKEN BRC mouse resources as a tool for analysis of gene function

○Kazuyuki Mekada,Fumio Ike,Noriko Hiraiwa,Hatsumi Nakata, Shinya Ayabe, Ayumi Murakami,Masayo Kadota,Megumi Tanaka,Maiko Ijyuin, Kyuichi Taguma, Keiji Mochida,Atsuo Ogura,Atsushi Yoshiki RIKEN BioResource Center, Tsukuba, Japan

The major goal of the RIKEN BRC experimental animal division is to collect, preserve and distribute valuable mice strains created in Japan. By February 2014, 7,239 mice models are collected, of which mice with a high demand are maintained as live animals, the other mice are preserved as frozen stock. Genetically modified strains especially with C57BL/6 background have been effectively preserved as frozen sperm. Collected mice were cleaned up by using cesarean section and embryo transfer, and maintained as SPF mice. We examined the genetically modified mice for their multiple transgenes to confirm their genetic quality. We have distributed our mouse resources (over 20,000 items) to about 900 domestic/overseas users, resulting in over 400 outstanding papers. Our mice have been distributed as live animals, frozen embryos/sperm and recovered litters from frozen stocks. RIKEN BRC has also participated in the IMPC, which aims to analyze the primary phenotypes of knockout mice by an internationally agreed standard pipeline and provide the mice with their penotyping data to researchers. We started to generate genome-wide knockout mice that meet emerging research needs, in collaborated with the Japan Mouce Clinic. These mice and data will be available to the research community in the near future.

S 174 3P086-S Intra-specific variation of hibernation-specific traits in Tamias sibiricus

○Taito Kamata,Ai Sakamoto,Tsuneo Sekijima

Graduate School of Science and Technology, University of Niigata, Niigata, Japan

Generally, it is well known that mammalian hibernators reduce energy consumption by hypothermia and hypometabolisim in winter. Regulatory mechanism of hibernation, however, has not been yet completely understood. Hibernation-specific Protein (HP), which was discovered from the blood of a chipmunk Tamias sibiricus, provided a great understanding in hibernation mechanism. During hibernation, HP in the blood was decreased and it became active form in the process transferring from the blood to the brain. Furthermore, successful artificial hibernation by suppression of Brain HP with HP antibody strengthened importance of HP in hibernation regulation. In the process of elucidating hibernation mechanism, we found out existence of non-hibernator as intra-specific variation in chipmunk. If non-hibernator was derived from variation of hibernation mechanism, chipmunk has a significant value as model animal of hibernation. In this study, we first established criteria to classify hibernator and non-hibernator in chipmunk. Then, we compared HP regulation traits in blood between two haplotypes. As a result, classification of either hibernator or non-hibernator was allowed by 258 days under monitoring condition by constant darkness and 5°C. As for HP regulation, hibernator had clearly annual rhythmicity, while non-hibernator had no rhythmicity. In conclusion, this study demonstrated the existence of hibernation polymorphisms derived from differential HP regulation.

3P087-S Possibility of artificial crossing inTamias sibiricus

○Ai Sakamoto,Taito Kamata,Tsuneo Sekijima

Graduate School of science and Technology, University of Niigata, Niigata, Japan

In the process of clarifying hibernation mechanism, we found out the existence of non-hibernator as well as hibernator as intra-specific variation of Tamias sibiricus, which would be useful to find out hibernation regulatory factor. In order to push for a genetic approach using this polymorphism of hibernation style, it is necessarily to establish a system of artificial crossing in this species. However, no successful breeding of this species under experimental condition has been reported so far. Our purposes in this study are to clarify estrus cycle in female, to identify a key signal leading to copulation success, and to implement artificial crossing of this species. In all monitored females, morphological change in female's genital regions was observed once a year. During estrus period, two types of estrus call were identified, but there was no significant relationship between differential estrus call and hibernation style. Compared copulatory behavior during vocalizing estrus call with that during not vocalizing estrus call, frequency of copulation was significantly higher during vocalizing than that during not vocalizing. In mating experiments of two haplotypes, successful copulation and gestation were obtained in all combination of inbreeding and outbreeding. In summary, hibernation style and estrus call did not disturb the artificial crossing in Tamias sibiricus. Also, estrus call may be a key factor succeeding in artificial crossing.

S 175 3P088-S Primary screening of diabetes animals from the closed colony of European wood mice

○Sayuri Tomiyama,Goro Kato,Hiroki Shichijo,Shinsuke Sakamoto, Akio Shinohara, Chihiro Koshimoto FSRC,University of Miyazaki,Miyazaki,Japan

AimEuropean wood mice (Apodemus sylvaticus) have been maintained as closed colony in University of Miyazaki to study its spontaneous hyperlipidemia to be a new animal model. In the process of this project, onsets of diabetes were found in some of these animals with/without hyperlipidemia. It is necessary and reasonable to separate these two different phenotypes to establish animal model. In this study, efficiency of primary screening was evaluated by polyposia as a simple index.Materials and methodsFood and water consumption, fecal and urine volumes, and body mass changes of 36 animals were recorded for 5 days by using metabolic cages. They were then divided into normal and polydipsia groups. The distributions of fasting blood glucose and total cholesterol levels were compared; the thresholds of diabetes and hyperlipidemia were 126mg /dl and 220mg/dl, respectively. We also measured their water consumption in regular cages.Results and discussionThe incidence of diabetes in polyposia (20ml water consumption/day or more, in metabolic cages) group was higher than the incidence in normal group (81% vs. 27%).These incidences ware similar when water consumption was recorded in regular cages and divided into two groups by the threshold of 45ml water consumption/2days (75% vs. 25%). The results suggest that the primary screening of diabetes animals from our colony can be applicable by water consumption, not only in metabolic cages but in regular cages also.

3P089-S Distribution project of gene-targeting mice in Tokyo University of Science

○Sachiko Kubo,Tomonori Kaifu,Mariko Tateishi,Yoichiro Iwakura

Center for Animal Disease Models, Research Institute for Biomedical Sciences, Tokyo University of Science, Chiba, Japan

[Purpose]Gene-targeting mice are recognized as an important bio-tool for dissecting physiological roles of the interested genes . Building a bio-resource system that delivers the bio-materials to researchers remarkably contributes to the progress of basic medicine and life science. We had provided a great number of original gene-targeting mice in the University of Tokyo, and since 2012, the bio-resource project of distributing bio-materials continue in Tokyo University of Science (TUS). Here, we demonstrate the status quo for distributing the bio-materials in TUS. [Method]A database of available gene-targeting mice was established and made public on the internet (http://www.rs.tus.ac.jp/iwakuralab/support.html). We prepared Materials Transfer Agreement, developed the co-operative relationship with TUS, and had an expert staff for this bio-resource project. [Results and Discussion]We distributed 1,396 bio-materials (641 for domestic academia, 604 for international; 44 for domestic company, 17 for international) until 2011 in the University of Tokyo. We have so far delivered 83 bio-materials in TUS, and there are a great number of distribution on IL-17s-, IL-1s-, and C-type lectin receptors-relating mice, reflecting that these mice are useful animal tools mainly in immunological study.

S 176