Production of a Homozygous Mutant Embryonic Stem Cell Line (Double Knockout)

Richard Mortensen1

1 University of Michigan Medical School, Ann Arbor, Michigan
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 23.6
DOI:  10.1002/0471142727.mb2306s82
Online Posting Date:  April, 2008
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Abstract

Under some circumstances, it may be desirable to produce a mouse cell clone in which both alleles of a desired gene are mutated. This may be because the mutation causes embryonic lethality in homozygous animals, or to test cultured cells before an animal is produced. This protocol details an easy method for obtaining homozygous cells by homologous recombination without the need for two targeting events. Curr. Protoc. Mol. Biol. 82:23.6.1‐23.6.4. © 2008 by John Wiley & Sons, Inc.

Keywords: homologous recombination; mouse; homozygous; mutation; double knockout

     
 
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Table of Contents

  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1:

  Materials
  • Heterozygous mutant ES cell line, frozen in liquid nitrogen (unit 23.5)
  • ES/LIF medium (unit 23.5)
  • G418 (unit 9.5)
  • 100‐mm tissue culture plates, gelatin coated (unit 23.5)
  • Additional reagents and equipment for recovery of frozen cell lines (unit 11.9), ES cell culture (units 23.2& 23.3& 3.NaN), northern analysis (unit 4.9), and immunoblotting (unit 10.8)
NOTE: All tissue culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise noted.
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Figures

Videos

Literature Cited

Literature Cited
   Mortensen, R.M., Conner, D.A., Chao, S., Geisterfer, L.A., and Seidman, J.G. 1992. Production of homozygous mutant ES cells with a single targeting construct. Mol. Cell. Biol. 12: 2391‐2395.
   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.
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