Backcrossing to an Appropriate Genetic Background Improves the Birth Rate of Carbohydrate Sulfotransferase 14 Gene-Deleted Mice

Experimental Animals Exp. Anim. 69(4), 407–413, 2020 Original Backcrossing to an appropriate genetic background improves the birth rate of carbohydrate sulfotransferase 14 gene-deleted mice Shin SHIMADA1), Takahiro YOSHIZAWA1), Yuki TAKAHASHI2), Yuko NITAHARA-KASAHARA3), Takashi OKADA3,4), Yoshihiro NOMURA5), Hitoki YAMANAKA1), Tomoki KOSHO2,6,7) and Kiyoshi MAtsUMOTO1) 1)Division of Animal Research, Research Center for Supports to Advanced Science, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan 2)Center for Medical Genetics, Shinshu University Hospital, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan 3)Department of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan 4)Division of Molecular and Medical Genetics, Center for Gene and Cell Therapy, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan 5)Scleroprotein and Leather Research Institute, Tokyo University of Agriculture and Technology, Faculty of Agriculture, 3-5-8 Saiwaicho, Fuchu, Tokyo 183-0057, Japan 6)Department of Medical Genetics, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan 7)Research Center for Supports to Advanced Science, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan Abstract: Ehlers–Danlos syndromes (EDSs) are heterogeneous group of heritable connective tissue disorders characterized by joint and skin hyperextensibility as well as fragility of various organs. Recently, we described a new type of EDS, musculocontractual EDS (mcEDS-CHST14), caused by pathogenic variants of the carbohydrate sulfotransferase 14 (CHST14) gene mutation. B6;129S5-Chst14tm1Lex/Mmucd (B6;129-Chst14 KO) mice are expected to be an animal model of mcEDS-CHST14. However, >90% of B6;129-Chst14 KO homozygous (B6;129- Chst14−/−) mice show perinatal lethality. Therefore, improvement of the birth rate of Chst14−/− mice is needed to clarify the pathophysiology of mcEDS-CHST14 using this animal model. Some B6;129-Chst14−/− embryos had survived at embryonic day 18.5 in utero, suggesting that problems with delivery and/or childcare may cause perinatal lethality. However, in vitro fertilization and egg transfer did not improve the birth rate of the mice. A recent report showed that backcrossing to C57BL/6 strain induces perinatal death of all Chst14−/− mice, suggesting that genetic background influences the birthrate of these mice. In the present study, we performed backcrossing of B6;129- Chst14 KO mice to a BALB/c strain, an inbred strain that shows lower risks of litter loss than C57BL/6 strain. Upon backcrossing 1 to 12 times, the birth rate of Chst14−/− mice was improved with a birth rate of 6.12–18.64%. These results suggest that the genetic background influences the birth rate ofChst14 −/− mice. BALB/c congenic Chst14−/− (BALB.Chst14−/−) mice may facilitate investigation of mcEDS-CHST14. Furthermore, backcrossing to an appropriate strain may contribute to optimizing animal experiments. Key words: backcrossing, birth rate, carbohydrate sulfotransferase 14 (Chst14), Ehlers-Danlos syndrome, genetic background (Received 29 November 2019 / Accepted 13 May 2020 / Published online in J-STAGE 10 June 2020) Corresponding author: T. Yoshizawa. e-mail: tyoshizawa@shinshu-u.ac.jp Supplementary Figures and Tables: refer to J-STAGE: https://www.jstage.jst.go.jp/browse/expanim This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License <http://creativecommons.org/licenses/by-nc-nd/4.0/>. ©2020 Japanese Association for Laboratory Animal Science S. SHIMADA, ET AL. Introduction Materials and Methods Ehlers–Danlos syndromes (EDSs) are a heterogeneous Animals group of heritable connective tissue disorders character- Chst14 KO mice were obtained from the Mutant ized by joint and skin hyperextensibility as well as fragil- Mouse Regional Resource Center (https://www.mmrrc. ity of various organs [3–5, 9, 10, 15, 19]. Recently, a org) and inbred for more than 12 generations [18]. Mice new type of EDS was found, which is caused by carbo- were housed under SPF conditions with a constant tem- hydrate sulfotransferase 14 (CHST14) gene mutations perature (23 ± 2°C), relative humidity (45–70%), and and named musculocontractural EDS (mcEDS-CHST14) 12-h light/dark cycle. Animals had free access to tap [7, 11]. The pathophysiological mechanisms and thera- water and standard mouse chow (Oriental Yeast Co., pies of mcEDS-CHST14 are not yet established. There- Ltd., Tokyo, Japan). fore, development of disease model animals is indispens- Based on the national regulations and guidelines, all able. experimental procedures were reviewed by the Commit- The B6;129-Chst14 KO mouse generated by Bian et tee for Animal Experiments of Shinshu University and al. [2] is expected to be a model of the pathophysiology finally approved by the President of Shinshu University of mcEDS-CHST14. However, perinatal lethality of (Approval numbers 280116, 280039, and 280038). All Chst14−/− mice has made investigations difficult. A pre- experimental procedures were carried out in accordance vious study showed that the genotypes of Chst14−/− em- with the Regulations for Animal Experimentation of bryos follow an approximate Mendelian distribution Shinshu University. (25%) until embryonic day (E) 16.5 [1]. On E18.5 just before delivery, some embryos survive (14.8%) in utero IVF-ET [18]. However, only a limited number of adult B6;129- Female mice with heterozygous Chst14 gene deletion Chst14−/− mice were produced (1.3%) [18]. These reports (Chst14+/−) were superovulated by intraperitoneal injec- suggest that problems with delivery and/or childcare may tions of 7.5 IU pregnant mare serum gonadotropin (Nip- affect the reproductive efficiency. pon Zenyaku Kogyo Co., Ltd., Fukushima, Japan) fol- To improve the birth rate, in vitro fertilization (IVF)- lowed by 7.5 IU human chorionic gonadotropin (hCG; embryo transfer (ET) is widely used for mouse strains Nippon Zenyaku Kogyo Co., Ltd.) after 48 h. At 13–15 with a poor birth rate [6]. The birth rate is different h after injection of hCG, female Chst14+/− mice were among some inbred mouse strains [12]. Akyuz et al. sacrificed humanely. Then, cumulus oocyte complexes reported that backcrossing B6;129-Chst14 KO mice were collected and placed in human tubal fluid (HTF) several times to the C57BL/6 strain results in perinatal medium (Ark Resource, Co., Ltd., Kumamoto, Japan) death of all Chst14−/− mice [1]. The BALB/c strain, a under mineral oil (Sigma-Aldrich Japan, Tokyo, Japan). common inbred mouse strain that is widely used for Sperm was collected from the cauda epididymis of male animal experiments, shows lower risks of litter loss than Chst14+/− mice and incubated for 1 h in HTF medium. the C57BL/6 strain [17]. Therefore, we considered that The cumulus oocyte complexes and sperm were cocul- −/− the birth rate of Chst14 mice might be influenced by tured at 37°C with 5% CO2 for 5–6 h. Fertilized ova were the genetic background. placed in modified human tubal fluid (mHTF) medium Inbred strain mice have almost the same genetic back- (Kyudo. Co., Ltd., Saga, Japan) and checked for cleavage ground. Generally, more than 12 backcrossings are nec- the next morning. Embryos (2-cell) were then transferred essary to generate a congenic strain. It is known that the to a fresh drop of mHTF medium and transplanted into repeat number of microsatellites is different among pseudopregnant ICR mice. To generate pseudopregnant mouse strains, which is used to identify the genetic back- recipients, unstimulated ICR female mice (Japan SLC, ground. A database of mouse microsatellites has been Shizuoka, Japan) were mated with vasectomized ICR provided by the National Institute of Genetics (Shizuo- male mice (Japan SLC) overnight. The female mice were ka, Japan) [14]. then checked for vaginal plugs. Days when plugs were In the present study, to improve the birth rate of found were designated as E0.5 of pseudopregnancy. At Chst14−/− mice, we performed IVF-ET and backcrossing E0.5 of pseudopregnancy, the embryos (2-cell) were to the BALB/c strain and checked the genetic back- transplanted into fallopian tubes (10 embryos/oviduct). ground of the mice by microsatellite markers. The genotypes of fetus (E18.5) and offspring were checked by polymerase chain reaction (PCR). A schema of the method for IVF-ET is shown in Supplementary Fig. 1A. 408 | doi: 10.1538/expanim.19-0150 GENETIC BACKGROUND AND BIRTH RATE Backcrossing of the microsatellite markers was set to be >10 centim- B6;129-Chst14 KO mice were backcrossed to the organs. When it was difficult to a set different PCR- BALB/cAJcl (BALB/cA) strain (CLEA Japan, Inc. Shi- product size of each of the three strains, the markers were zuoka, Japan) 12 times. First, female Chst14+/− and male set between two strains on the intergenic distance BALB/cA mice were mated. The progeny (F1) were (D4Mit254, D8Mit121, and D16Mit182 were used for genotyped to select Chst14+/− mice, and then Chst14+/− the BALB/c vs. C57BL/6 comparison; D4Mit225, F1 mice were used for backcrossing again with BALB/ D8Mit52, and D16Mit33 were used for the BALB/c vs. cA mice (Supplementary Fig. 1B). The F1 female and 129 comparison). The primer sequences are listed in male Chst14+/− mice were mated to examine the birth Supplementary Tables 1 and 2. PCR of the microsatellite rate of Chst14−/− mice (Supplementary Fig. 1C). This markers was carried out using the GoTaq® PCR Core procedure was repeated 12 times to generate the BALB/c System I (Promega K.K., Tokyo, Japan). The PCR con- congenic Chst14 KO (BALB.Chst14 KO) strain. To re- ditions were carried out according to the protocol of the place the sex (Y) chromosome and mitochondrial DNA, Mouse Microsatellite Database of Japan [14]. PCR prod- males and females were inverted and mated at the sev- ucts were visualized by electrophoresis with 2% agarose enth generation.

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