Generation of Transgenic Mice

Andrew Cho1, Naoto Haruyama1, Ashok B. Kulkarni1

1 National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 19.11
DOI:  10.1002/0471143030.cb1911s42
Online Posting Date:  March, 2009
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Abstract

This unit describes detailed step‐by‐step protocols, reagents, and equipment required for successful generation of transgenic mice using pronuclear injection. The experimental methods and practical tips given here will help guide beginners in understanding what is required and what to avoid in these standard protocols for efficiently generating transgenic mice. Curr. Protoc. Cell Biol. 42:19.11.1‐19.11.22. © 2009 by John Wiley & Sons, Inc.

Keywords: DNA purification; genotyping; embryo harvesting; embryo implantation; transgenic mice; zygote

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Purification of Transgenic Construct using a Sucrose Gradient
  • Alternate Protocol 1: Purification of Transgenic Construct using a Gel‐Based DNA Purification Method
  • Basic Protocol 2: Harvesting Donor Zygotes (Fertilized Eggs) for Microinjection
  • Basic Protocol 3: Microinjection of the Transgene DNA Construct into Mouse Zygote
  • Basic Protocol 4: Implantation of Microinjected Zygotes into Pseudo‐Pregnant Recipient Mice
  • Basic Protocol 5: Preparation of Tissue Samples for Genotyping Founder Mice
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Purification of Transgenic Construct using a Sucrose Gradient

  Materials
  • Plasmid DNA of interest, digested to remove plasmid backbone
  • TE buffer, pH 7.4 (KD medical)
  • 10% (w/v) and 40% (w/v) sucrose solutions (see recipes)
  • 1.2% agarose gel
  • Microinjection buffer or BAC DNA buffer (see recipes)
  • Sucrose gradient DNA purification apparatus (Fig. ) containing:
    • Custom‐made sucrose mixer
    • Stirrer/hot plate
    • Glass microcapillary (Kimble Glass)
    • Rubber tubing
    • Magnetic bar
    • 14 × 95–mm centrifuge tubes (Beckman)
  • Ultracentrifuge (e.g., Beckman Coulter, model L‐70)
  • Swinging‐bucket rotors (e.g., Beckman Coulter, model SW 40 Ti)
  • 1.7‐ml microcentrifuge tubes
  • Microcapillary pipet (Kimble Glass, 20‐µl)
  • Dialysis cassette (Pierce, 10,000 MWCO)
  • Additional reagents and equipment for checking the DNA concentration using a spectrophotometer ( appendix 3D)

Alternate Protocol 1: Purification of Transgenic Construct using a Gel‐Based DNA Purification Method

  • Transgene DNA of interest, digested to remove plasmid backbone (see protocol 1)
  • QIAEX II Gel Extraction Kit (QIAGEN, cat. no. 20051) including the following components:
    • Buffer QX1
    • QIAEX II DNA binding particles
    • Buffer PE
  • Razor blade
  • 50°C water bath
  • Vortex

Basic Protocol 2: Harvesting Donor Zygotes (Fertilized Eggs) for Microinjection

  Materials
  • PMSG (pregnant mare's serum gonadotropin; Sigma Chemical Inc., cat. no. G4877‐2000IU)
  • 20 donor female mice (4‐ to 5‐week‐old FVB/N mice)
  • HCG (human chorionic gonadotropin; Sigma Chemical Inc., cat. no. CG10‐1VL)
  • 20 stud male mice (sexually mature male, 2‐ to 12‐month‐old, FVB/N mice)
  • M16 medium (Sigma Chemical, cat. no. M7292)
  • M2 medium (Sigma Chemical, cat. no. M7167)
  • Hyaluronidase (Sigma Chemical, cat. no. H3884‐1G)
  • 35 × 10–mm tissue culture dish (Falcon, cat. no. 353001)
  • 60 × 10–mm tissue culture dish (Falcon, cat. no. 353002)
  • Dissecting microscope for handling zygotes (Zeiss, Stemi SV II Apo)
  • Light source for dissecting microscope (Zeiss, KL 1500 LCD)
  • Surgical scissors (Roboz, RS‐5840)
  • Microdissection forceps (Roboz, RS‐5055)
  • Mouth‐controlled pipet assembly (Fig. ) including the following items:
    • Plastic mouth piece
    • Portion of a 1‐ml pipet that includes cotton piece
    • Rubber tubing, 1/8‐in. i.d., 1/32‐in. wall thickness (Thomas Scientific, cat. no. 9521R67)
    • Glass tubing to make embryo‐handling pipets (Drummond Scientific, cat. no. 9‐000‐1181)
    • Glass tubing with a rubber cap (Drummond Scientific, cat. no. 1‐000‐0300)
  • Bunsen burner
  • CO 2 incubator (Thermo Electron Corporation, Model 370)
  • Additional reagents and equipment for peritoneal injections (Donovan and Brown, ) and euthanasia of mice (Donovan and Brown, )

Basic Protocol 3: Microinjection of the Transgene DNA Construct into Mouse Zygote

  Materials
  • M2 medium (Sigma Chemical, cat. no. M7167)
  • Mineral oil (Sigma Chemical, cat. no. M3516‐1L)
  • Washed, ready‐to‐inject zygotes ( protocol 3)
  • M16 medium (Sigma Chemical, cat. no. M7292)
  • Microforge (Technical Products International Corporation, MF‐1)
  • Microinjection system including the following components (Fig. A):
    • Inverted microscope (Zeiss, Axiovert 135M)
    • Anti‐vibration table (Kinetic systems)
    • Automated DNA injector (Eppendorf, Femptojet)
    • Capillary holder for holding pipet (Mitutoyo)
    • Micromanipulator (Narishige, model MO‐202U)
    • Compressed Nitrogen gas cylinder
  • Needle/pipet puller (Kopf Instruments, model 720)
  • Glass tubing for microinjection needles (Sutter Instrument, cat. no. BF100‐78‐10)
  • Microloaders (Eppendorf, cat. no. 930001007)
  • One‐chambered plastic slide to be used for the microinjection platform (Nalge Nunc International, cat. no. 177372)
  • Mouth‐controlled pipet assembly for handling zygotes (Fig. )
  • CO 2 incubator (Thermo Electron Corporation, model 370)

Basic Protocol 4: Implantation of Microinjected Zygotes into Pseudo‐Pregnant Recipient Mice

  Materials
  • Forty wild‐type female mice (CB6F1, 6‐ to 8‐weeks‐old) to be mated with vasectomized mice to generate pseudo‐pregnant recipient mice
  • Twenty vasectomized BALB/c mice (housed in separate cages)
  • M16 dish containing microinjected zygotes (see protocol 4)
  • 70% ethanol
  • 0.25% (v/v) Avertin for anesthesia
  • Alcohol swabs
  • Betadine antibiotic swabs
  • 0.5% (v/v) Marcaine for analgesia
  • Animal cages
  • CO 2 incubator (Thermo Electron Corporation, model 370)
  • Glass bead sterilizer for the surgical instruments (Fine Science Tools, type 250)
  • Dissecting microscope for transferring zygotes (Zeiss, Zeiss Stemi SV II Apo)
  • Electric clippers
  • Surgical instruments (Sigma Surgical Instrument) including:
    • Forceps
    • Scissors
    • Clamp skin closer with wound clips
    • Sutures
    • Curved forceps
  • Mouth‐controlled pipet assembly (Fig. )
  • Inverted microscope to perform surgery (Zeiss, Stemi 2000)
  • Light source to illuminate the surgical area (Zeiss, KL 1500 LCD)
  • Slide warmer preheated to 37°C (Lab‐line Instruments, model 26005)
  • Additional reagents and equipment for anesthetizing the mouse (Donovan and Brown, ) and intraperitoneal injections (Donovan and Brown, )

Basic Protocol 5: Preparation of Tissue Samples for Genotyping Founder Mice

  Materials
  • Potential founder mice
  • Tail lysis buffer (see recipe)
  • Proteinase K
  • Phenol
  • Chloroform
  • 100% and 70% ethanol
  • 1× TE buffer
  • Microtattoo system (Fine Science Tools, cat. no. 24201‐00)
  • Green tattoo paste (Fine Science Tools, cat. no. 24201‐01)
  • 30‐G needle (Becton Dickinson, cat. no. 305106)
  • Labeled microcentrifuge tubes
  • Hybridization oven (Boekel, Little Shot II, Model 230501)
  • Additional reagents and equipment for tail DNA isolation for Southern blotting (Brown, ) and PCR ( appendix 3F)
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Figures

  •   FigureFigure 19.11.1 Purification of a transgenic construct using the sucrose gradient method. (A) Sucrose mixer with dimensions of 3‐in. (W) × 2‐in. (H) × 1 7/8‐in. (D) is made by making two chambers of 5/8‐in. (D) × 2‐in. (H), which are connected together on the bottom with an outlet and two spigots that open and close controlling the sucrose flow into the ultracentrifuge tube. (B, C). A picture and schematic drawing showing how gradients are made from 10% to 40% sucrose.
  •   FigureFigure 19.11.2 Schematic diagram of the components necessary for preparing a mouth‐controlled pipet device for handling zygotes. Rubber tubing with 1/8‐in. i.d. and 1/32‐in. wall is cut in three pieces, and the mouth piece, 0.2‐µm syringe filter unit, 1‐ml pipet cotton piece, and glass tubing are attached to each end of rubber tubing.
  •   FigureFigure 19.11.3 Sequence of embryo harvesting: (A) Two M16, three M2, and one M2 medium with hyaluronidase plates are prepared for handling or culturing embryos throughout the whole process of transgenic mice generation. (B, C) The reproductive organs: uterus, ovary, and oviduct are removed from the donor mice. (D) All the oviducts are collected in a single M2 dish. (E) The ampulla is located and torn apart with forceps under the microscope. (F) The zygotes and the cumulus cells are spilled into the M2 medium with hyaluronidase. (G) The zygotes are slowly separated from the surrounding cumulus cells. (H) Thoroughly washed zygotes are ready for microinjection.
  •   FigureFigure 19.11.4 Making of a holding pipet. (A) Microforge. (B) The schematic diagram of preparing holding pipets. The hand‐pulled and diamond pencil‐cut straight‐edged pipet is selected and is brought closer to the heated glass ball to melt evenly to give a polished holding pipet with a small opening of ∼20 µm.
  •   FigureFigure 19.11.5 A schematic diagram for preparing injection needles. A glass tubing of 1.0‐mm o.d. × 0.78‐mm i.d. is inserted between two rubber pads and is pulled downward with the setting of 2.5 (downward force) × 16 (heat intensity) to prepare excellent and reproducible injection needles.
  •   FigureFigure 19.11.6 Microinjection setup. (A) A microinjection system. (B) Schematic diagram of the microinjection setup. A holding pipet is attached to the oil‐pressured controller on the left side of the microscope whereas the injection pipet (needle) is attached to the automatic microinjector on the right side.
  •   FigureFigure 19.11.7 A schematic diagram depicting microinjection of transgenic DNA into the pronucleus. The size of the microinjected male pronucleus expands as the DNA is injected. A quick horizontal injection maneuver while minimizing damage to the zygote is critical for successful gene delivery.
  •   FigureFigure 19.11.8 A sequence of events for the microinjection technique. (A) Injection platform for microinjection. Note the rectangular M2 medium in the center that is overlaid with mineral oil. (B) A polished holding pipet is lowered onto the injection platform and a small volume of M2 medium is sucked into the pipet. (C) A zygote is held in place by the holding pipet and the injection needle is lowered and the tip is focused. (D) DNA solution is injected into the male pronucleus. Note the swelling of the pronucleus, which confirms the successful transfer of the transgene.
  •   FigureFigure 19.11.9 A schematic diagram of preparing transfer pipets for zygote transfers. A hand‐pulled and diamond pencil‐cut straight‐edged pipet with ∼100‐µm opening size is first loaded with the five alternate sequence of M16 medium and small bubbles and followed by fifteen to twenty tightly packed zygotes.
  •   FigureFigure 19.11.10 A sequence of events for zygote transfer into oviduct. (A) A surgical site is located and shaved. (B) A small skin incision is made and the ovary and fat pad are located before the muscle membrane incision is made. (C) The ovary and an oviduct are pulled out by the fat pad with the small metal clip. (D) The bursa is carefully peeled off and tucked under the ovary with a pair of forceps. (E) The space between the ovary and oviduct is opened with a pair of forceps while the tip of another forceps is inserted into the opening of the oviduct called the infundibulum for locating and testing prior to transferring zygotes with transfer pipet. (F, G) The transfer pipet is inserted into the infundibulum and the whole embryo content is blown slowly into the oviduct. (H) The muscle membrane is sutured and one drop of analgesia is administered to relieve pain during recovery. (I) the recipient mouse is placed on the slide warmer at 37°C.
  •   FigureFigure 19.11.11 A schematic diagram depicting zygote transfer into the infundibulum, which is often hidden within coils of oviduct. The infundibulum can be made visible by peeling off the bursa membrane that covers the ovary and by separating the space between the ovary and the oviduct using fine forceps.

Videos

Literature Cited

   Brinster, R.L., Chen, H.Y., Trumbauer, M., Senear, A.W., Warren, R., and Palmiter, R.D. 1981. Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell 27:223‐231.
   Brown, T. 1993. Southern Blotting. Curr. Protoc. Molec. Biol. 21:2.9.1‐2.9.20.
   Conner, D.A. 2004. Transgenic mouse production by zygote injection. Curr Protoc. Molec. Biol. 68:23.9.1‐23.9.23.
   Donovan, J. and Brown, P. 2006a. Parenteral injections. Curr. Protoc. Immunol. 73:1.6.1‐1.6.10.
   Donovan, J. and Brown, P. 2006b. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
   Donovan, J. and Brown, P. 1998. Anesthesia. Curr. Protoc. Immunol. 27:1.4.1‐1.4.5.
   Giraldo, P. and Montoliu, L. 2001. Size matters: Use of YACs, BACs and PACs in transgenic animals. Transgenic Res. 10:83‐103.
   Jahner, D., Haase, K., Mulligan, R., and Jaenisch, R. 1985. Insertion of the bacterial gpt gene into the germ line of mice by retroviral infection. Proc. Natl. Acad. Sci. U.S.A. 82:6927‐6931.
   Lois, C., Hong, E.J., Pease, S., Brown, E.J., and Baltimore, D. 2002. Germline transmission and tissue‐specific expression of transgenes delivered by lentiviral vectors. Science 295:868‐872.
   Muyrers, J.P., Zhang, Y., Testa, G., and Stewart, A.F. 1999. Rapid modification of bacterial artificial chromosomes by ET‐recombination. Nucleic Acids Res. 27:1555‐1557.
   Schedl, A., Larin, Z., Mountoliu, L., Thies, E., Kelsey, G., Lerach, H., and Schutz, G. 1993. A method for the generation of YAC transgenic mice by pronuclear microinjection. Nucleic Acids Res. 21:4783‐4787.
   Sternberg, N.L. 1992. Cloning high molecular weight DNA fragments by the bacteriophage P1 system. Trends Genet. 8:11‐16.
   Testa, G., Vintersten, K., Zhang, Y., Benes, V., Muyrers, J.P., and Stewart, A.F. 2004. BAC engineering for the generation of ES cell‐targeting constructs and mouse transgenes. Methods Mol. Biol. 256:123‐139.
   van der Putten, H., Botteri, F.M., Miller, A.D., Rosenfeld, M.G., Fan, H., Evans, R.M., and Verma, I.M. 1985. Efficient insertion of genes into the mouse germ line via retroviral vectors. Proc. Natl. Acad. Sci. U.S.A. 82:6148‐6152.
   Wagner, T. E., Hoppe, P.C., Jollick, J.D., Scholl, D.R., Hodinka, R.L., and Gault, J.B. 1981. Microinjection of a rabbit beta‐globin gene in zygotes and its subsequent expression in adult mice and their offspring. Proc. Natl. Acad. Sci. U.S.A. 78:6376‐6380.
   Zhang, Y., Buchholz, F., Muyrers, J.P., and Stewart, A.F. 1998. A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 20:123‐128.
   Zhang, Y., Muyrers, J.P., Testa, G., and Stewart, A.F. 2000. DNA cloning by homologous recombination in Escherichia coli. Nat. Biotechnol. 18:1314‐1317.
Key References
   Brown, G.A.J. and Corbin, T.J. 2002. Transgenesis in the Mouse: Oocyte injection. In Transgenesis Techniques. Methods in Molecular Biology, Vol. 180 (A.R. Clarke, Ed.) pp. 39‐70. Humana Press, Totowa, New Jersey.
  This chapter contains very useful information for the production of transgenic mice, especially for setting up a new transgenic facility.
   Conner, 2004. See above.
  The most current information and methodologies for the generation of transgenic mice are found in this protocol.
   Nagy, A., Gertsenstein, M., Vintersten, K., and Behringer, R. 2003. Manipulating the Mouse Embryo: A laboratory Manual, 3rd Edition, Cold Spring Laboratory Press, Cold Spring Harbor, New York.
  A comprehensive guide for generating genetically altered mice along with excellent diagrams and illustrations.
   Overbeek, P.A. 1994. Factors Affecting Transgenic Animal Production In Transgenic Animal Technology: A Laboratory Handbook (Pinkert, C.A. Ed.) pp. 72‐109. Academic Press, San Diego.
  A detailed description of different strains of mice, excellent husbandry practices, and transgenic phenomenology are discussed in this chapter.
   Pinkert, C.A. 1994. Introduction to Transgenic Animals In Transgenic Animal Technology: A Laboratory Handbook (Pinkert, C.A. Ed.) pp. 3‐11. Academic Press, San Diego.
  A thorough and complete history of the development of transgenic technology, as well as a compendium for practical applications of transgenesis via pronuclear injections.
   Pinkert, C.A. and Polites, H.G 1994. Transgenic Animal Production Focusing on the Mouse Model In Transgenic Animal Technology: A Laboratory Handbook (Pinkert, C.A. Ed.) pp. 15‐65. Academic Press, San Diego.
  Another excellent resource for the production of transgenic animals via pronuclear microinjections.
   Testa et al., 2004. See above.
  An excellent guide for designing specific BAC transgenic constructs.
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