Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout)

Richard Mortensen1

1 University of Michigan Medical School, Ann Arbor, Michigan
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 23.5
DOI:  10.1002/0471142727.mb2305s82
Online Posting Date:  April, 2008
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Gene targeting by homologous recombination is a powerful and widely used technique for introduction of specific gene mutations (frequently a gene inactivation) in transgenic animals. The basic method detailed in this unit uses sequences homologous to the endogenous gene flanking the mutation. While methods using bacterial artificial chromosomes (BACs) and recombineering may be used, in most cases simpler bacterial plasmid clones with several kb of homology are sufficient. This protocol details the strategic factors in designing the constructs for selection and screening for homologous recombination. Curr. Protoc. Mol. Biol. 82:23.5.1‐23.5.11. © 2008 by John Wiley & Sons, Inc.

Keywords: homologous recombination; heterozygous mouse; mutation; single knockout

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Gene Targeting in Embryonic Stem Cells
  • Support Protocol 1: Transient Expression of CRE for Recombination
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Gene Targeting in Embryonic Stem Cells

  • Target gene from genomic library isogenic with ES cell line (e.g., 129 SV library; Stratagene)
  • Plasmid vector (e.g., pNTK, available from R. Mortensen; see Fig. )
  • 95% ethanol
  • Sterile H 2O
  • Embryonic stem (ES) cells (units 23.2& 23.3; ATCC)
  • ES/LIF medium (see recipe)
  • Trypsin/EDTA: 0.25% (w/v) trypsin/1 mM EDTA (20 mM HEPES, pH 7.3, optional)
  • ES medium (see recipe)
  • Electroporation buffer (see recipe)
  • G418 (unit 9.5)
  • Gancyclovir (GANC)
  • Freezing medium (see recipe)
  • Digestion buffer (see recipe)
  • Saturated NaCl (see recipe)
  • 1% agarose gel (unit 2.5)
  • Tissue culture hood
  • Gelatin‐coated tissue culture plates (unit 23.3): 100‐mm plates and 24‐well microtiter plates
  • 4‐mm electroporation cuvettes
  • Pipet tips, sterilized by autoclaving
  • 55°C temperature block or incubator
  • Nylon membrane
  • Additional reagents and equipment for subcloning DNA (unit 3.16), restriction enzyme digestion (unit 3.1), phenol/chloroform extraction of DNA (unit 2.1), agarose gel electrophoresis (unit 2.5), ES cell culture (units 23.2& 23.3 and 3.NaN), electroporation (unit 9.3), stable transformation using selective medium (unit 9.5), DNA quantitation ( 3.NaN), and Southern blotting and hybridization (units 2.9& 2.10)
NOTE: All tissue culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise noted.

Support Protocol 1: Transient Expression of CRE for Recombination

  • Cre expression plasmid using a promoter giving high expression levels in ES cells (e.g., pMC1 or pPGK)
  • 12.5 mg/ml 5‐fluorocytosine (to select against CD) in sterile PBS ( appendix 22)
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Literature Cited

Literature Cited
   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.
   Capecchi, M.R. 1989. Altering the genome by homologous recombination. Science 244: 1288‐1292.
   Cheng, S., Fockler, C., Barnes, W., and Higuchi, R. 1994. Effective amplification of long targets from cloned inserts and human genomic DNA. Proc. Natl. Acad. Sci. U.S.A. 91: 5695‐5699.
   Deng, C. and Capecchi, M.R. 1992. Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol. Cell. Biol. 12: 3365‐3371.
   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 microinjected into cultured mammalian cells: Evidence for homologous recombination between injected plasmid DNA molecules. Mol. Cell. Biol. 2: 1372‐1387.
   Hasty, P., Rivera, P.J., and Bradley, A. 1991. The length of homology required for gene targeting in embryonic stem cells. Mol. Cell. Biol. 11: 5586‐5591.
   Koller, B.H., Kim, H.‐S., Latour, A.M., Brigman, K., Boucher, R.C. Jr., Scambler, P., Wainwright, B., and Smithies, O. 1991. Toward an animal model of cystic fibrosis: Targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 88: 10730‐10734.
   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.
   Milstone, D.S., Bradwin, G., and Mortensen, R.M. 1999. Simultaneous Cre catalyzed recombination of two alleles to restore neomycin sensitivity and facilitate homozygous mutations. Nucleic Acids Res. 27: e10.
   Robertson, E.J. 1987. Embryo derived stem cell lines. In Teratocarcinomas and Embryonic Stem Cells: A Practical Approach (E.J. Robertson, ed.) pp. 71‐112. IRL Press, Oxford and New York.
   Smithies, O., Gregg, R.G., Boggs, S.S., Koralewski, M.A., and Kucherlapati, R.S. 1985. Insertion of DNA sequences into the human chromosomal beta‐globin locus by homologous recombination. Nature 317: 230‐234.
   teRiele, H., Maandag, E.R., and Berns, A. 1992. Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs. Proc. Natl. Acad. Sci. U.S.A. 89: 5128‐5132.
   Thomas, K.R., Deng, C., and Capecchi, M.R. 1992. High‐fidelity gene targeting in embryonic stem cells by using sequence replacement vectors. Mol. Cell. Biol. 12: 2919‐2923.
   Wong, E.A. and Capecchi, M.R. 1987. Homologous recombination between coinjected DNA sequences peaks in early to mid‐S phase. Mol. Cell. Biol. 7: 2294‐2295.
   Yenofsky, R.L., Fine, M., and Pellow, J.W. 1990. A mutant neomycin phosphotransferase II gene reduces the resistance of transformants to antibiotic selection pressure. Proc. Natl. Acad. Sci. U.S.A. 87: 3435‐3439.
   Zheng, H. and Wilson, J.H. 1990. Gene targeting in normal and amplified cell lines. Nature 344: 170‐173.
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