Generation of Gene Knockout Mice by ES Cell Microinjection

Glenn Longenecker1, 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 19.14
DOI:  10.1002/0471143030.cb1914s44
Online Posting Date:  September, 2009
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Abstract

This unit describes protocols used in the production of chimeric mice that are then used for the generation of gene knockout mice. These protocols include the collection of blastocyst embryos, ES cell injection, and uterine transfer of injected blastocysts. Support protocols for the superovulation of blastocyst donor mice, generation of pseudopregnant recipients, fabrication of glass pipets for embryo and cell manipulations, and generation of germline mice are also included. Practical tips and solutions are mentioned to help troubleshoot problems that may occur. Curr. Protoc. Cell Biol. 44:19.14.1.‐19.14.36. © 2009 by John Wiley & Sons, Inc.

Keywords: embryonic stem cells; blastocyst; blastocyst injection; uterine transfer; chimeric mice; knockout mice

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Collection of Blastocysts
  • Support Protocol 1: Superovulation of Embryo Donor Females
  • Support Protocol 2: Fabrication of Glass Pipets
  • Basic Protocol 2: Injection of Blastocysts with ES Cells
  • Basic Protocol 3: Uterine Transfer of Injected Blastocysts
  • Support Protocol 3: Generation of Pseudopregnant Recipient Females
  • Basic Protocol 4: Generation of Germline Knockout Mice
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Collection of Blastocysts

  Materials
  • C57BL/6NCr 3.5‐day post‐coitum (p.c.) superovulated female mice (see protocol 2; 10 are needed)
  • Injection medium (see recipe)
  • 70% ethanol
  • Microdrop cultures (see recipe)
  • Bench paper (absorbent material with plastic backing)
  • 35 × 10–mm tissue culture dishes (Falcon 3001 or equivalent)
  • 3‐cc tuberculin syringes
  • 30‐G, 0.5‐in. needles (Becton Dickson, cat. no. 305106 or equivalent)
  • 70% ethanol pads
  • Surgical instruments (cleaned and sterilized with alcohol pads):
    • Dissection scissors (Roboz RS‐5880 or equivalent)
    • No. 55 forceps (Roboz RS‐5063 or equivalent)
  • Dissecting microscope
  • Transfer pipet (see protocol 3)
  • Mouth pipet assembly (see Fig. 19.11.2) including:
    • Mouthpiece (Fisher Scientific no. 13‐647‐10 or equivalent)
    • Saliva trap (cotton plug from a 1‐mm pipet; BD Falcon, cat. no. 7521, or equivalent)
    • Syringe filter, 0.22‐µm (Gelman no. 4602 or equivalent)
    • Pipet insert and reservoir (Drummond microcaps no. 1‐000‐0300 or equivalent); a modified 1000‐µl pipet tip can be used as a reservoir.
    • Approximately 23 in. of natural latex tubing (1/8‐in. i.d.; 1/32‐in. wall)
    • Male luer, 5/32‐in. (Ark‐Plas Products no. 10‐12ML016N or equivalent) to connect female end of syringe filter to rubber tubing
    • Female luer, 5/32‐in. (Ark‐Plas Products no. 10‐15FL016N or equivalent) to connect male end of syringe filter to rubber tubing
  • Additional reagents and equipment for euthanasia of the mouse (Donovan and Brown, )

Support Protocol 1: Superovulation of Embryo Donor Females

  Materials
  • C57Bl/6NCr females or equivalent strain (3 to 4 weeks of age; ten are needed per injection day
  • 50 IU/ml pregnant mare's serum gonadotrophin (PMSG; see recipe)
  • 50 IU/ml human chorionic gonadotrophin (HCG; see recipe)
  • 10 C57Bl/6NCr males (7 weeks to 10 months of age)
  • 1‐cc tuberculin syringes
  • 30‐G, 0.5‐in. needles (Becton Dickson no. 305106 or equivalent)
  • Additional reagents and equipment for injection of mice (Donovan and Brown, )

Support Protocol 2: Fabrication of Glass Pipets

  Materials
  • Plasticene (Fisher Scientific, cat. no. P148‐1LB or equivalent)
  • 1.25% Tween 80 solution (see recipe)
  • Glass tubing (Drummond Scientific no. N‐51A: I.D. 0.8 mm × O.D. 1.0 mm × length 150 mm or equivalent)
  • Microflame assembly (for pulling transfer pipets and polishing ends of pipets, consisting of:
    • Rectangular support stand plus rod (PGC Scientific, or equivalent)
    • Alumaloy tri‐grip utility buret clamps with micro clamp holder (PGC Scientific, or equivalent)
    • 9‐in. Pasteur pipet shortened to 7 in. (clamped vertically to the support stand, using the buret clamps, to emit a small flame)
    • Tygon tubing, formulation R‐3603 (I.D. 1/4 in. × O.D. 3/8 in. × wall 1/16 in.) for conveying gas to the Pasteur pipet
  • Diamond pencil
  • Microforge (Defonbrune style, with 10× eye pieces and reticle, 4× and 10× objectives or equivalent; Technical Products International, http://www.nuhsbaum.com); glass bead added to platinum wire filament in the laboratory with pulled injection or holding pipet
  • Instrument holder to hold pipets (Leica, cat. no. 520145 or equivalent)
  • C‐flex tubing: I.D. 1/32 in. × O.D. 3/32 in. × wall 1/32 in. × length ∼4 mm long (Cole Parmer, cat. no.6424‐60 or equivalent) to hold pipets inside instrument holder
  • 150 × 25–mm tissue culture dish (Falcon 3025 tissue culture plate or equivalent)
  • Pipet puller (Kopf 1720 vertical pipet puller with nichrome heater coil or equivalent)
  • Top of 100 × 20–mm tissue culture dish (Falcon 3010 tissue culture plate or equivalent)
  • Dissecting microscope (10×/23 eye pieces, 0.6‐6.6× zoom, with base illumination)
  • 1‐cc tuberculin syringe
  • 30‐G, 0.5 in. needle (Becton Dickson, cat. no. 305106 or equivalent)
  • 35 × 10–mm tissue culture dishes (BD Falcon, cat. no. 3001, or equivalent)
  • Syringe pipet assembly (to coat injection pipets with 1.25% Tween 80 solution) including:
    • 12‐cc tuberculin syringe
    • Female luer 1/8 in. (Ark‐Plas Products no. 10‐15FL012N or equivalent) to connect syringe with tubing
    • Pipet insert and reservoir (Drummond microcaps no. 1‐000‐0300 or equivalent); a modified 1000‐µl pipet tip may also be used as a reservoir.
    • Natural latex tubing (I.D. 1/8 in. × wall 1/32 in. × ∼8 in. long)

Basic Protocol 2: Injection of Blastocysts with ES Cells

  Materials
  • Dulbecco's phosphate‐buffered saline (DPBS) without calcium and magnesium (Mediatech, cat. no. 21‐031‐CV, or equivalent)
  • Liquid soap (e.g., VioNex; any liquid soap will probably work)
  • Injection medium (see recipe)
  • ES cell suspension (1 × 106/ml) transformed with desired transgene (unit 19.13)
  • Filtered light white mineral oil (Sigma M‐3516 embryo‐tested or equivalent)
  • Blastocysts (see protocol 1)
  • 60 × 15–mm tissue culture plate (Falcon 3002 or equivalent)
  • 70% ethanol pads
  • Injection chamber with cooling system (Fig. )
    • Water circulating pumps (Baxter, cat. no. 8329 or equivalent)
    • Siliconized tubing, I.D. 3/16‐in., with plastic connections
    • Male luers with 1/16 in. barb (Cole Parmer, cat. no. 31507‐62 or equivalent) to connect plastic connections of silicone tubing to inflow and outflow Tygon tubing
    • Injection dish (see Fig. )
    • Tygon tubing for inflow and outflow cooling lines, I.D. 3/32 in. × O.D. 5/32 in. × wall 1/32 in.
    • 60 cc tuberculin syringe with Tygon tubing to load water jacket of injection dish
    • Female luer with 3/16 in. to 1/4 in. barb (Cole Parmer, cat. no. 31507‐62 or equivalent) connected to inflow tubing as a weight in ice water
  • Transfer pipet, cleaned and polished ( protocol 3)
  • Mouth pipet assembly (see Fig. 19.11.2 and protocol 1)
  • Dissecting microscope (10×/23 eye pieces, 1.0× objective, 0.6 to 6.6× zoom, with base illumination)
  • Injection pipet (see protocol 2)
  • Holding pipet (see protocol 2)
  • Microinjection setup (see recipe and see Fig. ) consisting of:
    • Injection apparatus:
    • Inverted microscope (Zeiss 135M or equivalent)
    • Coarse micromanipulators (microscope‐mounted; Narishige MMN‐1 or equivalent)
    • Fine micromanipulators (Joy‐stick micromanipulator; Narishige MO‐202U or equivalent)
    • Mounting bracket for mounting Narishige course manipulators
    • Video camera (Dage‐MTI CCD‐72 or equivalent)
    • Video monitor (Sony PVM‐137 or equivalent)
    • Injection line:
    • Instrument holder to hold injection pipet (Leica, cat no.520145 or equivalent)
    • Tygon tubing AAC0001: I.D. 1/16 in. × O.D. 1/8 in. × wall 1/32 in. × ∼4‐mm long tubing insert for instrument holder to connect injection pipet to PE 60 tubing
    • Polyethylene tubing for injection line (Becton Dickinson PE 60, no. 427415, I.D. 0.030 in. × O.D. 0.048 in. × length ∼2 ft. or equivalent)
    • Tygon tubing AAC0001: I.D. 1/16 in. × O.D. 1/8 in. × wall 1/32 in. × ∼8 mm long or equivalent to connect between Tygon tubing AAC0002 and PE 60 tubing
    • Tygon tubing AAC0002: I.D. 1/16 in. × O.D. 1/8 in. × wall 1/32 in. × ∼1 cm long or equivalent to connect Tygon AAC0001 tubing to PE 200 tubing
    • Polyethylene tubing for injection line (Becton Dickson PE 200; Clay Adams no. 427440, I.D. 0.055 in. × O.D. 0.075 in. or equivalent), ∼3.5 cm. in length
    • Female luer flare type adapter (Popper no. 6193 or equivalent) to connect PE 200 tubing to 3‐way stopcock
    • 3‐way stopcock (Baxter no. K177A or equivalent)
    • Tygon tubing (Cole Parmer no. 6408‐63, I.D. 3/32 in. × O.D. 5/32 in. × wall 1/32 in. or equivalent) for injection line between the stopcock and to either the syringe reservoir or the Hamilton syringe
    • Female luer with barb (BioRad, cat. no. 7318223 or equivalent) to connect Hamilton syringes to Tygon tubing
    • Male luer with barb (BioRad, cat. no. 7318226 or equivalent) to connect to luer lock connection of stopcock to Tygon tubing
    • Micrometer syringe (Minnetonka Instruments or equivalent) with Mitutoyo syringe, 0 to 50 mm, with 50‐µl gas‐tight syringe (Hamilton no. 80920 or equivalent for injection line)
    • Female luer connection with barb (Cole Parmer no. 31507‐65 or equivalent) to connect Cole Parmer 6408‐63 to tuberculin syringe reservoir
    • Tuberculin syringe (12 cc) containing Dow Corning silicone oil 200 for reservoir
    • Holding line:
    • Instrument holder to hold holding pipet (Leica, cat. no. 520145 or equivalent)
    • Tygon tubing AAC0001: I.D. 1/16 in. × O.D. 1/8 in. × wall 1/32 in. × length ∼4 mm or equivalent tubing insert for instrument holder to connect holding pipet to PE 60 tubing
    • Polyethylene tubing for holding line (Becton Dickinson PE 100; Clay Adams no. 427425, I.D. 0.034 in. × O.D. 0.06 in. × approximate length 2 ft., or equivalent)
    • Tygon tubing AAC0001: I.D. 1/16 in. × O.D. 1/8 in. × wall 1/32 in. × length ∼1.8 cm, or equivalent, to connect between PE 100 tubing and blunted 19‐G needle
    • Blunted 19‐G needle to connect male luer end of stopcock to Tygon AAC0001 tubing
    • 3‐way stopcock (Baxter, cat. no. K177A or equivalent)
    • Tygon tubing (Cole Parmer no. 6408‐63: I.D. 3/32 in. × O.D. 5/32 in. × wall 1/32 in., or equivalent) for injection line from the stopcock to either the syringe reservoir or the Hamilton syringe
    • Female luer with barb (BioRad, cat. no. 7318223 or equivalent) to connect Hamilton syringe to Tygon tubing
    • Male luer with barb (BioRad, cat. no. 7318226 or equivalent) to connect luer lock connection of stopcock to Tygon tubing
    • Micrometer syringe (Minnetonka instruments or equivalent) with Mitutoyo syringe, 0 to 50 mm with 250 µl gas‐tight syringe (Hamilton, cat. no. 81120 for holding line)
    • Female luer connection with barb (Cole Parmer, cat. no.31507‐65 or equivalent) to connect Cole Parmer 6408‐63 to tuberculin syringe reservoir
    • Tuberculin syringe (12 cc) containing silicone oil 200 for reservoir
    • Ice bucket containing ice and water
    • Air suspension platform setup:
    • Air suspension table (Kinetic Systems 9101‐02‐46 or equivalent)
    • Gas regulator (Curtis Mathis 1H‐580 or equivalent)
    • Nitrogen gas cylinder (Roberts Oxygen no. R‐31 or equivalent)

Basic Protocol 3: Uterine Transfer of Injected Blastocysts

  Materials
  • Injection medium (see recipe)
  • Filtered light white mineral oil (Sigma M‐3516 embryo tested or equivalent)
  • Injected blastocysts in microdrop cultures (see protocol 4)
  • 2.5‐day p.c. pseudopregnant B6D2F1 female mice or equivalent (see protocol 6)
  • 2.5% Avertin working solution (see recipe)
  • 0.5% Marcaine (Hospira, cat. no. NDC 0409‐1610‐50; http://www.hospira.com/) or 0.5% Naropin analgesic (AstraZeneca, cat. no. NDC0186‐0863‐61)
  • Transfer pipets (see protocol 3)
  • Mouth pipet assembly (see Fig. 19.11.2 and protocol 1)
  • 35 × 10–mm tissue culture dishes (BD Falcon, cat. no. 3001)
  • 100 × 20‐mm tissue culture dish (BD Falcon, cat. no. 3010) containing two Plasticene rolled strips (Fisher Scientific, cat. no. P148‐1LB or equivalent); do not sterilize prior to surgery
  • Dissecting microscope (10×/23 eye pieces, 1.0× objective, 0.6 to 6.6× zoom, with base illumination)
  • Mouse fur clippers (Oster finisher trimmer no. 76059‐030 or equivalent)
  • 70% ethanol pads
  • Betadine swabs
  • Surgical stand (6‐1/8 in. × 7‐1/2 in. Nalgene container top with vinyl bumpers attached to bottom, or equivalent)
  • Surgical microscope (16×/16 eye pieces, 0.63× objective, 0.8 to 6.6× zoom) with Diagnostics SMS6B sliding zoom stand or equivalent)
  • Fiber‐optic light source (Zeiss KL‐1500 or equivalent)
  • Surgical instruments, cleaned and sterilized by placing in glass bead sterilizer (Fine Science Tools FTS 250 or equivalent) for 10 sec prior to use in surgery:
    • Dissection scissors (Roboz RS‐5880 or equivalent)
    • No. 7 curved forceps (Roboz RS‐5064 or equivalent)
    • Foerster forceps, straight tips (Roboz RS‐5065 or equivalent)
    • Dieffenbach clamp (Roboz RS 7422 or equivalent)
    • Wound clip–removing forceps (Roboz RS‐9268 or equivalent)
    • Wound clip applier, Reflex 9 (Roboz RS‐9260 or equivalent)
    • 9‐mm wound clips (Roboz 9262 or equivalent)
  • Autoclaved sterilization pouch 5‐1/4 in. × 10 in. (Fisher Scientific, cat. no. 01‐812‐54 or equivalent) to serve as sterile field for instruments
  • Sterile gauze, 2 × 2‐in. and 4 × 4‐in.
  • 25‐G, 5/8‐in. needles
  • 1‐cc tuberculin syringes
  • Sterile cotton swabs
  • 37°C slide warmer
  • Additional reagents and equipment for injection of the mouse (Donovan and Brown, )

Support Protocol 3: Generation of Pseudopregnant Recipient Females

  Materials
  • B6D2F1 (C57Bl/6NCr x DBA/2NCr) female mice, or equivalent strain (10 to 12 weeks of age)
  • Balb/cAnNCr vasectomized male mice, or equivalent strain (at least 6 to 7 weeks to 18 months of age)
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Figures

Videos

Literature Cited

   Bradley, A. 1993. Production and analysis of chimeric mice. In Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. (E.J. Robertson, ed.) pp. 113‐151. Oxford University Press, New York.
   Bradley, A., Evans, M., Kaufman, M.H., and Robertson, E. 1984. Formation of germ‐line chimaeras from embryo‐derived teratocarcinoma cell lines. Nature 309:255‐256.
   Brayton, C., Mähler, M., and Nicklas, W. 2004. Viral infections. In The Laboratory Mouse. (H. Hedrich, ed.) pp. 357‐391. Elsevier Academic Press, New York.
   Brinster, R.L. 1974. The effects of cells transferred into the mouse blastocyst on subsequent development. J. Exp. Med. 140:1049‐1056.
   Doetschman, T. 1999. Interpretation of phenotype in genetically engineered mice. Lab. Anim. Sci. 49:137‐143.
   Doetschman, T., Maeda, N., and Smithies, O. 1988. Targeted mutation of the Hprt gene in mouse embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 85:8583‐8587.
   Donovan, J. and Brown, P. 2006a. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
   Donovan, J. and Brown, P. 2006b. Parenteral injections. Curr. Protoc. Immunol. 73:1.6.1‐1.6.10.
   Evans, M.J. and Kaufman, M.H. 1981. Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154‐156.
   Folger, K.R., Wong, E.A., Wahl, G., and Capecchi, M.R. 1982. Patterns of integration of DNA microinjection into cultured mammalian cells: Evidence for homologous recombination between injected plasmid DNA molecules. Mol. Cell. Biol. 2:1372‐1387.
   Gardner, R.L. 1968. Mouse chimeras obtained by the injection of cells into the blastocyst. Nature 220:596‐597.
   Guan, K., Nayernia, K., Maier, L.S., Wagner, S., Dressel, R., Lee, J.H., Nolte, J., Wolf, F., Li, M., Engel, W., and Hasenfuss, G. 2006. Pluripotency of spermatogonial stem cells from adult mouse testis. Nature 440:1199‐1203.
   Hogan, B., Beddington, R., Constantini, F., and Lacy, E. 1994. Manipulating the Mouse Embryo: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Hooper, M., Hardy, K., Handyside, A., Hunter, S., and Monk, M. 1987. HPRT‐deficient (Lesch‐Nyhan) mouse embryos derived from germ line colonization by cultured cells. Nature 326:292‐295.
   Kahan, B.W. and Ephrussi, B. 1970. Developmental potentialities of clonal in vitro cultures of mouse testicular teratoma. J. Natl. Cancer Inst. 44:1015‐1036.
   Khillan, J.S. and Bao, Y. 1997. Preparation of animals with a high degree of chimerism by one‐step coculture of embryonic stem cells and preimplanation embryos. Biotechniques 22:544‐549.
   Koller, B.H., Hagemann, L.J., Doetschman, T., Hagaman, J.R., Huang, S., Williams, P.J., First, N.L., Maeda, N., and Smithies, O. 1989. Germ‐line transmission of a planned alteration made in a hypoxanthine phosphoribosyltransferase gene by homologous recombination in embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 86:8927‐8931.
   Kuehn, M.R., Bradley, A., Robertson, E.J., and Evans, M.J. 1987. A potential animal model for Lesch‐Nyhan syndrome through introduction of HPRT mutations into mice. Nature 326:295‐298.
   Martin, G.R. 1981. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. U.S.A. 78:7634‐7638.
   McLaren, A. and Michie, D. 1956. Studies on the transfer of fertilized mouse eggs to uterine foster‐mothers: I. Factors affecting the implantation and survival of native and transferred eggs. J. Exp. Biol. 33:394‐416.
   McLaren, A. and Biggers, J.D. 1958. Successful development and birth of mice cultivated in vitro as early embryos. Nature 182:877‐878.
   Nagy, A., Rossant, J., Nagy, R., Abramow‐Newerly, W., and Roder, J.C. 1993. Derivation of completely cell culture‐derived mice from early‐passage embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 90:8424‐8428.
   Papaioannou, V.E. and Rossant, J. 1983. Effects of the embryonic environment on proliferation and differentiation of embryonal carcinoma cells. Cancer Surveys 2:165‐183.
   Papaioannou, V. and Johnson, R. 2002. Production of chimeras by blastocyst and morula injection of targeted ES cells. In Gene Targeting: A Practical Approach, 2nd ed. (A.L. Joyner, ed.) pp. 133‐175. Oxford University Press, New York.
   Rosenthal, M.D., Wishnow, R.M., and Sato, G.H. 1970. In vitro differentiation of clonal populations of multipotent mouse cells derived form a transplantable testicular teratocarcinoma. J. Natl. Cancer Inst. 44:1001‐1014.
   Samuel, K., Clarke, A.R., Ansell, J.D., and Hooper, M.L. 1993. Age dependent selection against phosphoribosyl transferase–deficient cells in mouse haematopoiesis. Development 118:859‐863.
   Silver, L.M. 1995. Mouse Genetics: Concepts and Application. Oxford University Press, New York.
   Simpson, E.M., Linder, C.C., Sargent, E.E., Davisson, M.T., Mobraaten, L.E., and Sharp, J.J. 1997. Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice. Nat. Genet. 16:19‐27.
   Smithies, O., Gregg, R.G., Boggs, S.S., Koralewski, M.A., and Kucherlapati, R.S. 1985. Insertion of DNA sequences into the human chromosomal β‐globin locus by homologous recombination. Nature 317:230‐234.
   Stevens, L.C. 1970. The development of transplantable teratocarcinomas from intratesticular grafts of pre‐ and postimplantation mouse embryos. Dev. Biol. 21:364‐382.
   Stevens, L.C. 1973. A new inbred subline of mice (129/terSv) with a high incidence of spontaneous congenital testicular teratomas. J. Natl. Cancer Inst. 50:235‐242.
   Stewart, C.S. 1993. Production of chimeras between embryonic stem cells and embryos. In Guide to Techniques in Mouse Development. Methods in Enzymology, Vol. 225 (P.M. Wassarman and M.L. DePamphilis, eds.) pp. 823‐854. Academic Press, New York.
   Takenaka, M., Horiuchi, T., and Yanagimachi, R. 2007. Effects of light on development of mammalian zygotes. Proc. Natl. Acad. Sci. U.S.A. 104:14289‐14293.
   Thomas, K.R. and Capecchi, M.R. 1987. Site‐directed mutagenesis by gene targeting in mouse embryo‐derived stem cells. Cell 51:503‐512.
   Thompson, S., Clarke, A.R., Pow, A.M., Hooper, M.L., and Melton, D.W. 1989. Germ line transmission and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells. Cell 56:313‐321.
   Wells, D. 1993. Production of chimeras derived from murine embryonic stem cells. Methods Mol. Biol. 18:217‐237.
   Wood, S.A., Pascoe, W.S., Schmidt, C., Kemler, R., Evans, M.J., and Allen, N.D. 1993. Simple and efficient production of embryonic stem cell–embryo chimers by coculture. Proc. Natl. Acad. Sci. U.S.A. 90:4582‐4585.
   Wong, G.T. 2002. Speed congenics: Applications for transgenic and knock‐out mouse strains. Neuropeptides 36:230‐236.
Key References
   Hogan et al., 1994. See above.
  Good overall reference on mouse surgeries and injections.
   Robertson, E.J. 1993. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Oxford University Press, New York.
  This reference source is especially useful for working with ES cells and solving troubleshooting problems encountered during ES cell injections. Bradley et al. () in this compilation is particularly relevant to the subject matter of this unit.
   Joyner, A.L. 2000. Gene Targeting; A Practical Approach, 2nd ed. Oxford University Press, New York.
  Another good reference for the procedures related to the production of chimeric mice. Especially useful for information in construct design. Papaioannou and Johnson () in this compilation is particularly relevant to the subject matter in this unit.
   Wassarman, P.M. and DePamphilis, M.L. 1993. Guide to techniques in mouse development. Methods Enzymol. vol. 225.
  This reference is good in characterizing the best morphology of ES cells to inject. It also describes multiple versions of the same procedures. Stewart () in this compilation is particularly relevant to the subject matter of this unit.
Internet Resources
  http://www.biosupplynet.com/
  This is a useful Web site for finding products and reagents required for procedures in the generation of chimeric mice and related procedures.
  http://www.microinjectionworkshop.net
  This site is useful for links to other transgenic‐related Internet sites. There are also transgenic‐related protocols as well as images of these techniques.
  http://oacu.od.nih.gov/additional/survivalrodent.html
  Ordering page for Training in Survival Rodent Surgery CD‐ROM (2001), developed by a subcommittee of the NIH Animal Research Advisory Committee.
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