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 Neuroscience
Unit Number:  Unit 4.30
DOI:  10.1002/0471142301.ns0430s55
Online Posting Date:  April, 2011
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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. Neurosci. 55:4.30.1‐4.30.12. © 2011 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 (Conner, , b; 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 4.6)
  • Gancyclovir (GANC)
  • Freezing medium (see recipe)
  • Digestion buffer (see recipe)
  • Saturated NaCl (see recipe)
  • 1% agarose gel ( appendix 1N)
  • Tissue culture hood
  • Gelatin‐coated tissue culture plates (unit 1.1): 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 (Struhl, ), restriction enzyme digestion ( appendix 1M), phenol/chloroform extraction of DNA ( appendix 1G), agarose gel electrophoresis ( appendix 1N), ES cell culture (Conner, , b), electroporation ( appendix 1E), stable transformation using selective medium (unit 4.6), DNA quantitation ( appendix 1K), and Southern blotting and hybridization (Brown, , )
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 2A)
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Literature Cited

Literature Cited
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   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.
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