DOCSLIB.ORG

The Eppendorf Transferman® 4R, One Manipulator for All Genetic Engineering Techniques

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Eppendorf Transferman® 4R, One Manipulator for All Genetic Engineering Techniques APPLICATION NOTE No. 285 The Eppendorf TransferMan® 4r, one manipulator for all genetic engineering techniques Ronald Naumann, Transgenic Core Facility, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Abstract The study of genetically modified animals provides impor- mouse or rat models prefer to have two separate work- tant insights into gene function. It can also be used for stations for the two main techniques, nucleic acid injec- modelling human diseases, to either understand disease tion into fertilized oocytes and embryonic stem (ES) cell mechanisms or aid drug development. transfer into early embryos. The main techniques to generate those animal models With the multifunctional TransferMan 4r, one workstation require micromanipulation devices, but usually in slightly is adequate for all applications because of its easy angle varying set-ups and with different settings for the respec- adjustment, vibration-free movements and unique Eppen- tive techniques. Therefore many laboratories generating dorf DualSpeed™ joystick. Introduction Micromanipulation of oocytes or embryos is the method for modifying the genome of laboratory animals. The most com- mon method is microinjection of nucleic acid constructs like simple transgenes, larger bacterial artificial chromosomes (BAC), or site-specific nucleases like zinc finger (ZFN) and transcription activator-like effector nucleases (TALEN). In recent times, the microinjection of clustered regularly inter- spaced short palindromic repeats (CRISPR) and RNA-guided Cas9 nucleases emerged as a powerful tool for genome engineering. The different types of constructs are either injected into one of the pronuclei of early zygotes (1-2) or into the cytoplasm (3-4). These techniques are called pronuclear and cytoplas- mic injection, respectively. Another technique, though not applicable to all laboratory animals due to the lack of suit- able material and/or limited accessibility of material, is the so-called ES cell transfer, i.e. the injection of genetically modified embryonic stem cells into embryos in blastocyst Figure 1: Eppendorf TransferMan 4r micromanipulators. or 8-cell stage. In addition, the Intracytoplasmic Sperm Injection (ICSI) of either genetically modified sperms or sperms coated with nucleic acid (sperm-mediated gene transfer) into oocytes can be used to generate genetically altered offspring (5). APPLICATION NOTE I No. 285 I Page 2 In practice, all these injection techniques demand certain > Adapter for inverted microscope, Eppendorf (Germany) precautions and considerations for optimal results. They > CellTram 4r Air microinjector for holding the oocyte, do not only differ in the optimal injection angles, but (in Eppendorf (Germany) our opinion) also vary in their demand of the micromanipu- > CellTram 4r Oil microinjector for transferring the ES cells, lators used as well as in the speed of injection movements. Eppendorf (Germany) Therefore most laboratories are using independently > FemtoJet® microinjector for pronuclear and cytoplasmic pre-fixed and finely adjusted workstations for each kind injections, Eppendorf (Germany) of injection technology. > Holding capillaries, e.g. VacuTip I, Eppendorf (Germany) > Injection capillaries, e.g. BioMedical Instruments With the TransferMan 4r (see Figure 1) Eppendorf (Germany) introduces a multifunctional manipulator suitable for > ES cell transfer capillaries, e.g. TransferTip® ES, Piezo all these applications. Drill Tip ES, Eppendorf (Germany) > Eppendorf Microloader™ for back-filling the injection This system includes a number of special features includ- capillaries, Eppendorf (Germany) ing application-specific masks like »Cell transfer«, »DNA injection« or »ICSI« which facilitate and simplify the indi- vidual workflow process. The unique DualSpeed joystick allows precise and intuitive movement during injection in A all spatial dimensions as well as dynamic movements while e.g. collecting ES cells. Thanks to its mounting concept the workstation is sturdy and safe against external vibrations. Furthermore, all elements of the manipulator are easy and fast to install and adjust. The construction of the motor modules allows for an extremely flexible set-up and can be adapted to all major microscopes used. Position changes on the motor modules are extremely flexible and therefore comfortable for all microscopes. The angle adjustment is easily done. It is possible to set shallow angles of theoreti- cally 0°, preferred for pronuclear and cytoplasmic injections B when using straight capillaries, as well as angles up to 45° (with a maximum of 90°). We tested the TransferMan 4r performance for manipulation techniques which are routinely performed in our lab over an extended period of time. Where possible, this was done in parallel to our standard set-up. Materials and Methods Figure 2: Display of the TransferMan 4r control panel: A) Initial display: »Choose Your Application«; Devices B) »Cell transfer« application mask selected with function keys for position > Inverted microscope equipped with Differential Interference storage and Z-axis limit. The 4th softkey is user-definable e.g. for »Axial«, »Clean« or other functions. Contrast (DIC), and 10 x, 20 x and 40 x objectives > Infrared Laser for laser-assisted ES cell transfer, e.g. from OCTAX® (Germany) or Hamilton Thorne (USA) > Two TransferMan 4r micromanipulators (one for moving the holding capillary and the second for positioning the transfer/injection capillary), Eppendorf (Germany) APPLICATION NOTE I No. 285 I Page 3 Operation of the micromanipulator TransferMan 4r Furthermore, the DualSpeed joystick can be operated with Operating the TransferMan 4r micromanipulator with two different movement modes. The standard mode is a its functional softkeys and its single joystick for all move- direct, intuitive movement, a direct transfer of the hand ment dimensions and speed settings is extremely simple movement to the microcapillary. When the maximum (see Figure 2): The selection of the application mask »Cell deflection of the actual path radius has been reached it is transfer« allows for the saving and recall of three capillary possible to press the joystick gently against its outer margin positions, plus the setting of a vertical limit and thus the to activate the dynamic mode so that the needle proceeds avoidance of capillary breakage. The application mask straight in the desired direction with an accelerating speed »DNA injection« offers not only capillary position storage as long as the joystick is deflected (see Figure 3). Using this but also functions to temporarily deactivate the Y motor DualSpeed joystick system, the needle can be moved care- preventing accidental sideways tearing of the zygote during fully in the fine or extra fine (x-fine) speed mode for gentle the sensitive injection procedure. This feature can be of manipulation of cells and embryos whilst still being capable crucial importance especially when the user is not very of a considerable range of quick motion once the dynamic, experienced with this injection technique. The single joystick outer zone of the joystick range is entered. By using this provides easy, intuitive movement control of the micro- feature even cells which are located in the periphery of your capillary in any direction (X, Y, Z), and each TransferMan 4r working focus can be quickly reached and collected. The can store up to five independent positions (depending on the combination of both features, the user-definable application chosen application mask). By simply pressing the joystick mask and the DualSpeed joystick, speeds up the entire work key twice, the capillary can be moved to one of the next flow and therefore shortens the time of samples being held preprogrammed positions. under the light beam of the microscope. Preparation of cells, embryos and injection samples The preparatory steps are of crucial importance but their detailed description would go beyond the scope of this Application Note. They will therefore not be described here, please proceed as described elsewhere (1, 6). Many different organisms may be used for pronuclear and cytoplasmic injection, and there are also more and more laboratory models for the transfer of ES cells. Although the general procedure of both microinjection techniques is very similiar for the different species, this Application Note focuses on the generation of mouse models. Figure 3: Principle of the DualSpeed joystick with direct (1) and dynamic deflection (2). APPLICATION NOTE I No. 285 I Page 4 Preparation of the workstation for pronuclear There are different injection chambers available to serve as (and cytoplasmic) injection a microenvironment for the zygote during the injection pro- One TransferMan 4r micromanipulator is used for moving cess (e.g. plastic Petri dish with glass bottom, depression the holding capillary and the other is used for moving the slide injection chamber or metal frame/glass slide injection injection capillary (Figure 4A). chamber). In our lab, we use a homemade injection chamber A CellTram 4r Air microinjector in combination with a hold- according to our specification. One drop of M2 medium is ing capillary, e.g. the VacuTip I, is used to hold the embryo. placed in the chamber. The zygotes to be injected (10 to 20) The programmable microinjector FemtoJet is used together are placed in one area of this drop. The whole droplet is with finely tapered microinjection capillaries to inject the covered with oil. nucleic acids. The FemtoJet offers an automatic
Load more

Recommended publications
  • Genetically Modified Organisms: Activity 1 Page 1 of 3
    Genetically Modified Organisms: Activity 1 Page 1 of 3 Activity 1: Coming Attractions Based on video content 15 minutes (10 minutes before and 5 minutes after the video) Setup Before watching the video, answer a few quick questions about genetically modified organisms. Don’t think too long about the questions—just write down your first response. You might already know the answers to some of them; otherwise, just take an educated guess. During the video, listen for the topics addressed by the questions. After the video, review the questions and the answers as a group. Materials • Transparency of the Coming Attractions Questions (master copy provided) • Transparency of the Coming Attractions Answers (master copy provided) Rediscovering Biology - 323 - Appendix Genetically Modified Organisms: Activity 1 (Master Copy) Page 2 of 3 Coming Attractions Questions 1. What two U.S. crops are the most heavily genetically modified? 2. What fraction of U.S. corn is genetically engineered? 3. What percent of U.S. processed food contains some genetically modified corn or soybean product? 4. All known food allergies are caused by one type of molecule. Is it proteins, sugars, or fats? 5. What is the substance called “bt” used for? a. Bonus: What does “bt” stand for? b. Bonus: When was “bt” first registered with the USDA? 6. What does “totipotent” mean? 7. What nutrient is produced by “golden rice”? 8. According to the World Health Organization, how many children go blind each year from vitamin A deficiency? 9. What is the normal function of “anti-thrombin”? 10. For what was “Dolly the sheep” famous? 11.
    [Show full text]
  • Genetic Engineering: Moral Aspects and Control of Practice
    Journal of Assisted Reproduction and Genetics, VoL 14. No. 6, 1997 NEWS AND VIEWS REPRODUCTIVE HEALTH CARE POLICIES AROUND THE WORLD Genetic Engineering" Moral Aspects and Control of Practice INTRODUCTION outline the concept of gene therapy with regard to the ethical aspects involved. Since the time of Hippocrates, physicians have sought to understand the'mechanisms of disease development for the purposes of developing more HISTORY effective therapy. The application of molecular biol- ogy to human diseases has produced significant The following is a concise review of the history discoveries about some of these disease states. of gene therapy. Extensive reviews can be found Gene transfer involves the delivery to target cells elsewhere (1). The human species has for many of an expression cassette made up of one or more generations practiced genetic manipulation on genes and the sequence controlling their expres- other species. Examples can be seen in the breed- sion. The process is usually aided by a vector that ing of animals and plants for specific intent, such helps deliver the cassette to the intracellular site as, corn, roses, dogs, and horses. The term "genet- where it can function appropriately. Human gene ics" was first used in 1906, and the term "genetic therapy has moved from feasibility and safety stud-. engineering" originated in 1932. Genetic engi- ies to clinical application more rapidly than was neering was then defined as the application of expected. The development of recombinant DNA genetic principles to animal and plant breeding. technology brought science to the brink of curing The term "gene therapy" was adopted to distin- previously untreatable diseases in a relatively short guish itself from the ominous, germ-line perception period of time.
    [Show full text]
  • Microinjection Applications
    Introduction to Microinjection Single-cell microinjection is a powerful and versatile technique for introducing exogenous material into cells and for extracting and transferring cellular components between cells. Any microinjection system will typically comprise three basic functions: the imaging of the target cell and the micropipette, the positioning of the micropipette, and control of the pressure inside the micropipette. The XenoWorks™ Microinjection system has been devised to fulfill the require- ments for the last two elements and to be versatile enough to be used for most microinjection and micromanipulation applications on a number of different microscope platforms. General Considerations Workstation Effective, reproducible microinjection requires a suitable quiet location, away from sources of vibration (slamming doors, elevators, centrifuges etc.), fluctuating temperature, drafts and bright light. In addition, convenient access to facilities such as incubators, compressed air (for antivibration tables) and operating theaters needs to be considered where necessary. Limiting the amount of vibration to the microinjection workstation is vital. The measures which need to be taken will depend upon the amount of ambient vibration. In general, the workstation should be set up in a quiet room, with little or no foot traffic, no slamming doors or heavy machinery such as elevators, centrifuges and refrigerators. Select a heavy, sturdy table or bench with plenty of room surrounding it, and ensure that the workstation components, including trailing wires and tubes, are not in contact with the floor or walls. If vibration is still detected (this can be judged by projecting a micropipette into the microscope field of view under high magnification and noting the behavior of the micropipette tip), use of an antivibration table should be consid- ered.
    [Show full text]
  • High Efficiency Transient and Stable Transformation by Optimized DNA Microinjection Into Nicotiana Tabacum Protoplasts
    Journal of Journal of Experimental Botany, Vol. 46, No. 290, pp. 1157-1167, September 1995 Experimental Botany High efficiency transient and stable transformation by optimized DNA microinjection into Nicotiana tabacum protoplasts Benedikt Kost, Alessandro Galli, tngo Potrykus and Gunther Neuhaus1 Institute for Plant Sciences, Swiss Federal Institute of Technology, Universita'tsstrasse 2, CH-8092 ZOrich, Switzerland Received 13 February 1995; Accepted 18 May 1995 Abstract higher. Incubation of embedded protoplasts at 4°C before microinjection was found to reduce the per- An efficient system has been established that allows centage of resistant clones obtained per injected cell. well controlled DNA microinjection into tobacco Protoplasts were immobilized above a grid pattern {Nicotiana tabacum) mesophyll protoplasts with par- and the location of injected cells was recorded by tially regenerated cell walls and subsequent analysis Polaroid photography. The fate of particular targeted of transient as well as stable expression of injected cells could be observed. Isolation and individual cul- reporter genes in particular targeted cells or derived ture of clones derived from injected cells was possible. clones. The system represents an effective tool to Following cytoplasmic coinjection of FITC-dextran and study parameters important for the successful trans- 1 mg ml"1 plasmid DNA on average about 20% of the formation of plant cells by microinjection and other targeted cells developed into microcalli and roughly techniques. Protoplasts were immobilized in a very 50% of these calli were stably transformed. Transient thin layer of medium solidified with agarose or algin- expression of the firefly luciferase gene {Luc) was non- ate. DNA microinjection was routinely monitored by destructively analysed 24 h after injection of pAMLuc.
    [Show full text]
  • Biological Activity Ofcloned Retroviral DNA in Microinjected Cells
    Proc. Natl Acad. Sci. USA Vol. 78, No. 7, pp. 4383-4387, July 1981 Cell Biology Biological activity of cloned retroviral DNA in microinjected cells (long terminal repeat/transcription/promoter assay) JOHN J. KOPCHICK, GRACE Ju, A. M. SKALKA, AND DENNIS W. STACEY Roche Institute of Molecular Biology, Nutley, NewJersey 07110 Communicated by B. L. Horecker, April 1, 1981 ABSTRACT Avian retroviral DNA molecules that had been 2.2) (2). The LTR is believed to contain the promoter se- cloned from infected cells by using recombinant DNA techniques quence(s) for viral transcription (8, 9). were microinjected into either uninfected chicken embryo fibro- Because the enzyme Sal I used to clone the circular DNA blasts (CEF) or CEF transformed by the envelope glycoprotein- cleaves within the viral env gene (1, 2, 9), the cloned molecules deficient Bryan strain of Rous sarcoma virus [RSV(-) cells]. Ret- contained a permuted gene order with portions of env gene at roviral DNA injected into RSV(-) cells directed transcription of each terminus of the virus-specific region within the linear A envelope mRNA, which was then able to complement the RSV(-) vector (Fig. IA) (2). Therefore, the production of env mRNA env deficiency and promote the production of infectious trans- in a recipient cell would require (i) removal of virus-specific forming virus. The retroviral DNA also directed the production DNA from the A vector, (ii) ligation of the ends of the cloned of fully infectious virus after injection into uninfected cells or and RSV(-) cells. Virus production began within 3-4 hr after microin- DNA to form a circle or linear concatemer (Fig.
    [Show full text]
  • Plant Cell Microinjection Technique
    Europaisches Patentamt 0175 966 Jt European Patent Office Publication number: B1 Office europeen des brevets EUROPEAN PATENT SPECIFICATION A 01 H1/00, Date of publication of patent specification: 24.01.90 Intel.5: C 12 N 15/00, C12N 1/08 Application number: 85111126.0 Date of filing: 04.09.85 Plant cell microinjection technique. (30) Priority: 25.09.84 US 654606 Proprietor: CALGENE, INC. 1920 Fifth Street Suite F. Davis California 95616 (US) Date of publication of application: 02.04.86 Bulletin 86/14 Inventor: Crossway, Anne 1902 El Paso Avenue, No. 4 Publication of the grant of the patent: Davis California 95616 (US) 24.01.90 Bulletin 90/04 Inventor: Facciotti, Daniel 2636 Lafayette Davis California 95616 (US) Designated Contracting States: AT BE CH DE FR GB IT LI LU NL SE Representative: Glawe, Delfs, Moll & Partner Patentanwalte References cited: Postfach 26 01 62 Liebherrstrasse 20 WO-A-83/01176 D-8000 Miinchen 26 (DE) PROTOPLASMA, vol. 1 16, 1983 (Wien, 1 New York); H.-H.STEINBISS etal.: "Protoplast OQ Derived Tobacco Cells Can Survive Capillary Microinjection of the Fluorescent Dye Lucifer Yellow", 223-227 CO pp. CO SCIENCE, vol. 151, no. 3708, 21 Jan 1966, o WASHINGTON, DC (US); T.P.LIN: of 333-337 in "Microninjection mouse eggs", pp. Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall have been filed until the opposition fee has been a.
    [Show full text]
  • Application of Genetically Engineered Pigs in Biomedical Research
    G C A T T A C G G C A T genes Review Application of Genetically Engineered Pigs in Biomedical Research Magdalena Hryhorowicz 1,*, Daniel Lipi ´nski 1 , Szymon Hryhorowicz 2 , Agnieszka Nowak-Terpiłowska 1, Natalia Ryczek 1 and Joanna Zeyland 1 1 Department of Biochemistry and Biotechnology, Poznan University of Life Sciences, Dojazd 11, 60-632 Pozna´n,Poland; [email protected] (D.L.); [email protected] (A.N.-T.); [email protected] (N.R.); [email protected] (J.Z.) 2 Institute of Human Genetics, Polish Academy of Sciences, Strzeszy´nska32, 60-479 Pozna´n,Poland; [email protected] * Correspondence: [email protected] Received: 4 May 2020; Accepted: 17 June 2020; Published: 19 June 2020 Abstract: Progress in genetic engineering over the past few decades has made it possible to develop methods that have led to the production of transgenic animals. The development of transgenesis has created new directions in research and possibilities for its practical application. Generating transgenic animal species is not only aimed towards accelerating traditional breeding programs and improving animal health and the quality of animal products for consumption but can also be used in biomedicine. Animal studies are conducted to develop models used in gene function and regulation research and the genetic determinants of certain human diseases. Another direction of research, described in this review, focuses on the use of transgenic animals as a source of high-quality biopharmaceuticals, such as recombinant proteins. The further aspect discussed is the use of genetically modified animals as a source of cells, tissues, and organs for transplantation into human recipients, i.e., xenotransplantation.
    [Show full text]
  • Physical Methods for Genetic Plant Transformation
    Available online at www.sciencedirect.com Physics of Life Reviews 9 (2012) 308–345 www.elsevier.com/locate/plrev Review Physical methods for genetic plant transformation Ana Leonor Rivera a, Miguel Gómez-Lim b, Francisco Fernández a, Achim M. Loske a,∗ a Centro de Física Aplicada y Tecnología Avanzada, Universidad Nacional Autónoma de México, A.P. 1-1010, C.P. 76000, Querétaro, Qro., Mexico b Unidad de Biotecnología e Ingeniería Genética de Plantas, Centro de Investigación y de Estudios Avanzados del IPN, A.P. 629, C.P. 36500, Irapuato, Gto., Mexico Received 2 June 2012; accepted 4 June 2012 Available online 6 June 2012 Communicated by J. Fontanari Abstract Production of transgenic plants is a routine process for many crop species. Transgenes are introduced into plants to confer novel traits such as improved nutritional qualities, tolerance to pollutants, resistance to pathogens and for studies of plant metabolism. Nowadays, it is possible to insert genes from plants evolutionary distant from the host plant, as well as from fungi, viruses, bacteria and even animals. Genetic transformation requires penetration of the transgene through the plant cell wall, facilitated by biological or physical methods. The objective of this article is to review the state of the art of the physical methods used for genetic plant transformation and to describe the basic physics behind them. © 2012 Elsevier B.V. All rights reserved. Keywords: Genetic plant transformation; Electroporation; Biolistics; Silicon carbide fiber; Ultrasound; Shock waves Contents 1. Introduction . 309 2. Electroporation . 312 3. Biolistics: particle bombardment . ............................................. 318 4. Vacuuminfiltration.................................................................... 319 5. Ultrasound-mediated transformation . 320 6.
    [Show full text]
  • Transformation and Microinjection* §
    Transformation and microinjection* § Thomas C. Evans, ed. , Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, CO 80045 USA Table of Contents 1. Introduction ............................................................................................................................2 2. General considerations for DNA transformation ............................................................................ 2 2.1. Scoreable/selectable marker genes .................................................................................... 2 2.2. Transgene regulatory elements ......................................................................................... 2 3. Microinjection ......................................................................................................................... 3 3.1. Protocol 1. Microinjection ............................................................................................... 4 4. DNA transformation: Creating repetitive extrachromosomal arrays by microinjection ........................... 7 4.1. Introduction ................................................................................................................. 7 4.2. Protocol 2. Formation of repetitive arrays by microinjection .................................................. 7 5. DNA transformation: Creating complex arrays by microinjection ...................................................... 8 5.1. Introduction ................................................................................................................
    [Show full text]
  • The Case for Restricting Genetic Enhancement Research
    Volume 110 Issue 2 Dickinson Law Review - Volume 110, 2005-2006 10-1-2005 Creating a Perfect Human Is Not So Perfect: The Case for Restricting Genetic Enhancement Research Sarah M. Markwood Follow this and additional works at: https://ideas.dickinsonlaw.psu.edu/dlra Recommended Citation Sarah M. Markwood, Creating a Perfect Human Is Not So Perfect: The Case for Restricting Genetic Enhancement Research, 110 DICK. L. REV. 473 (2005). Available at: https://ideas.dickinsonlaw.psu.edu/dlra/vol110/iss2/7 This Comment is brought to you for free and open access by the Law Reviews at Dickinson Law IDEAS. It has been accepted for inclusion in Dickinson Law Review by an authorized editor of Dickinson Law IDEAS. For more information, please contact [email protected]. Creating a Perfect Human Is Not So Perfect: The Case for Restricting Genetic Enhancement Research Sarah M. Markwood* I. Introduction Code 46, a scientific thriller movie, presents a world where the human race is altered and shaped by genetic engineering procedures.' While this futuristic movie is presently enthralling audiences throughout the world, scientific advances are rapidly creating a world where genetic enhancement is actually possible.2 Imagine a scenario where a mother and father desire their firstborn child to be an athletically gifted son who excels in basketball as a power forward. The only dilemma is both parents are below average in height and have no athletic skills. Instead of adjusting their aspirations for this child, the parents are able to utilize a genetic enhancement procedure and choose the "perfect" genes for him. Now the child will grow to an ideal basketball height of 6'9", possess the quickness and agility to handle the ball, and have the astuteness to analyze each situation encountered on the basketball court and decide which play must be executed.
    [Show full text]
  • Embryo Microinjection and Electroporation in the Chordate Ciona Intestinalis
    Journal of Visualized Experiments www.jove.com Materials List for: Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis Willi Kari1, Fan Zeng1, Lena Zitzelsberger1, Johannes Will1, Ute Rothbächer1 1 Department of Evolution and Developmental Biology, Zoological Institute, University Innsbruck Correspondence to: Ute Rothbächer at [email protected] URL: https://www.jove.com/video/54313 DOI: doi:10.3791/54313 Materials Name Company Catalog Number Comments Lab with constant temp. (~18 °C) For embryo work Ciona intestinalis adults UPMC - Station Biologique, For collecting gametes Roscoff, France NaCl Roth 3957.1 For ASWH KCl Roth 6781.1 For ASWH CaCl2x2H2O Roth A119.1 For ASWH MgCl2x6H2O Roth 2189.1 For ASWH MgS04x7H2O Roth P027.2 For ASWH NaHCO3 Roth 6885.1 For ASWH Hepes Roth HN78.3 For ASWH Sodium thioglycolate Sigma MZ-6 For Dechorionation Pronase Sigma T0632-100G For Dechorionation D-Mannitol Sigma Aldrich M9546-250G For electroporation Plasmid DNA (prep>1µg/µl) Macherey-Nagel 740410.100 For electroporation or microinjection Agarose any supplier For coating embryo culture dishes Plastic Petri dishes VWR 612-2079 Coated with 1% Agarose in ASWH, covered with ASWH Small scissors any supplier For dissecting gametes NaOH Roth 6771.1 For activating dechorionation solution Tris Roth 5429.3 1 M pH 9.5, for sperm activation Pasteur pipettes VWR 612-1701 Treated by immersion in tap water for at least 12 hr Hand centrifuge Hettich Zentrifugen 1011 Use glass centrifuge tubes 1.5 ml Eppendorf tubes Sigma T3406-250EA
    [Show full text]
  • The Potential Applicability of Gene Transfer Technology to the Human
    Int. J. Med. Sci. 2004 1(2): 76-91 76 International Journal of Medical Sciences ISSN 1449-1907 www.medsci.org 2004 1(2):76-91 ©2004 Ivyspring International Publisher. All rights reserved Review/Analysis Gene Therapy: The Potential Applicability of Gene Received: 2004.3.24 Transfer Technology to the Human Germline Accepted: 2004.5.14 Published:2004.6.01 Kevin R. Smith School of Contemporary Sciences, University of Abertay, Dundee, DD1 1HG, United Kingdom Abstract The theoretical possibility of applying gene transfer methodologies to the human germline is explored. Transgenic methods for genetically manipulating embryos may in principle be applied to humans. In particular, microinjection of retroviral vector appears to hold the greatest promise, with transgenic primates already obtained from this approach. Sperm-mediated gene transfer offers potentially the easiest route to the human germline, however the requisite methodology is presently underdeveloped. Nuclear transfer (cloning) offers an alternative approach to germline genetic modification, however there are major health concerns associated with current nuclear transfer methods. It is concluded that human germline gene therapy remains for all practical purposes a future possibility that must await significant and important advances in gene transfer technology. Key words gene targeting; gene therapy; genetic modification; pronuclear microinjection; sperm-mediated gene transfer Author biography Kevin Smith lectures in genetics and physiology in the School of Contemporary Sciences, University of Abertay, Dundee, United Kingdom. His research interests include transgenesis, gene therapy and bioethics. He is an experienced review article writer and has published several excellent reviews in the fields of transgenesis, gene therapy and bioethics. Corresponding K.
    [Show full text]