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
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

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

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Gene Targeting in Embryonic Stem Cells

  Materials
  • 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)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •   FigureFigure 4.30.1 pNTK vector. Both the neo and TK genes are driven by a PGK promoter ( pPGK) that is expressed in ES cells. Unique restriction enzyme sites that are useful are indicated in bold. One genomic fragment can be cloned into the BamHI site. A second genomic fragment can be cloned into the HindIII, ClaI, SalI, XhoI sites. A site should be preserved that will linearize the construct, leaving the majority of plasmid vector sequences attached to the TK gene (e.g., XhoI).
  •   FigureFigure 4.30.2 Constructs containing loxP sites surrounding a positive selectable marker, neo (A), or both a positive and a negative selectable marker, neo and cytosine deaminase ( CD; B). Constructs can be made by insertion of homologous sequences in unique restriction sites outside the loxP sites. If conditional targeting constructs are desired (as in Mortensen, ; Fig. 23.1.7), a third loxP site can be inserted into the region of homology and then the two regions of homology inserted into the vectors. Another version of these plasmids is also available with the TK and CD reversed (Milstone et al., ).
  •   FigureFigure 4.30.3 Production, selection, and identification of targeted gene disruption by homologous recombination. An example of a restriction enzyme site (RE) and hybridization probe that can be used to identify cells in which homologous recombination has occurred (shaded colony) is shown. The predicted size of the restriction fragment generated from an unaltered target gene (E) and a target gene that has undergone homologous recombination (HR) is shown. If equal amounts of DNA are present in the lanes of the Southern blot, the intensity of each of the two hybridizing fragments from the DNA of a homologous recombinant clone will be half of the intensity of the hybridizing fragment from unaltered clones.
  •   FigureFigure 4.30.4 Two strategies to create a replacement construct. In method (A), the target gene fragment is subcloned into a plasmid vector, then pPGKneo is inserted into a rare restriction enzyme site in the target‐gene fragment and pPGK‐TK is inserted into the plasmid vector near the target gene. In method (B), the target‐gene fragment is cleaved into two pieces that are subcloned into the polylinker sites of pNTK (see Fig. ). Note that the relative orientation of homologous fragments in the construct must retain that found in the target gene.

Videos

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.
   Brown, T. 1993. Hybridization analysis of DNA blots. Curr. Protoc. Mol. Biol. 21:2.10.1‐2.10.16.
   Brown, T. 1999. Southern blotting. Curr. Protoc. Mol. Biol. 68:2.9.1‐2.9.20.
   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.
   Conner, D.A. 2000a. Mouse embryo fibroblast (MEF) feeder cell preparation. Curr. Protoc. Mol. Biol. 51:23.2.1‐23.2.7.
   Conner, D.A. 2000b. Mouse embryonic stem (ES) cell culture. Curr. Protoc. Mol. Biol. 51:23.3.1‐23.3.6.
   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.
   Edmondson, D.G. and Roth, S.Y. 2001. Identification of protein interactions by far western analysis. Curr. Protoc. Mol. Biol. 55:20.6.1‐20.6.10.
   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 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.
   Jeong, Y. and Epstein, D.J. 2005. Modification and production of BAC transgenes. Curr. Protoc. Mol. Biol. 71:23.11.1‐23.11.15.
   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.
   Kramer, M.F. and Coen, D.M. 2001. Enzymatic amplification of DNA by PCR: Standard procedures and optimization. Curr Protoc. Mol. Biol. 56:15.1.1‐15.1.14.
   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.
   Mortensen, R. 2006. Overview of gene targeting by homologous recombination. Curr. Protoc. Mol. Biol. 76:23.1.1‐23.1.12.
   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.
   Struhl, K. 1987. Subcloning of DNA fragments. Curr. Protoc. Mol. Biol. 13:3.16.1‐3.16.2.
   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.
   Thomason, L., Court, D.L., Bubunenko, M., Costantino, N., Wilson, H., Datta, S. and Oppenheim, A. 2007. Recombineering: Genetic engineering in bacteria using homologous recombination. Curr. Protoc. Mol. Biol. 78:1.16.1‐1.16.24.
   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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library