Overview of Gene Targeting by Homologous Recombination

Richard Mortensen1

1 University of Michigan Medical Center, Ann Arbor, Michigan
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 23.1
DOI:  10.1002/0471142727.mb2301s76
Online Posting Date:  November, 2006
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Formerly UNIT 9.15, this unit has been moved to the opening spot of our new chapter on Embryonic Stem Cell technology. The unit has also been updated, and now includes information about the Cre‐lox and FlP/FRT recombinase systems.

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Table of Contents

  • Source of DNA
  • Anatomy of Targeting Constructs
  • Methods of Enrichment for Homologous Recombinants
  • Types of Mutations
  • Cre/loxP System
  • Literature Cited
  • Figures
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Literature Cited

Literature Cited
   Abuin, A. and Bradley, A. 1996. Recycling selectable markers in mouse embryonic stem cells. Mol. Cell. Biol. 16:1851‐1856.
   Agah, R., Frenkel, P.A., French, B.A., Michael, L.H., Overbeek, P.A., and Schneider, M.D. 1997. Gene recombination in postmitotic cells. Targeted expression of Cre recombinase provokes cardiac‐restricted, site‐specific rearrangement in adult ventricular muscle in vivo. J. Clin. Invest. 100:169‐179.
   Awatramani, R., Soriano, P., Rodriguez, C., Mai, J.J., and Dymecki, S.M. 2003. Cryptic boundaries in roof plate and choroid plexus identified by intersectional gene activation. Nat. Genet. 35:70‐75.
   Branda, C.S. and Dymecki, S.M. 2004. Talking about a revolution: The impact of site‐specific recombinases on genetic analysis in mice. Dev. Cell 6:7‐28.
   Brocard, J., Warot, X., Wendling, O., Messaddeq, N., Vonesch, J.L., Chambon, P., and Metzger, D. 1997. Spatio‐temporally controlled site‐specific somatic mutagenesis in the mouse. Proc. Natl. Acad. Sci. U.S.A. 94:14559‐14563.
   Brocard, J., Feil, R., Chambon, P., and Metzger, D. 1998. A chimeric Cre recombinase inducible by synthetic, but not by natural ligands of the glucocorticoid receptor. Nucl. Acids Res. 26:4086‐4090.
   Copeland, N.G., Jenkins, N.A., and Cour, D.L. 2001. Recombineering: A powerful new tool for mouse functional genomics. Nat. Rev. Genet. 2:769‐779.
   Cruz, A., Coburn, C.M., and Beverley, S.M. 1991. Double targeted gene replacement for creating null mutants. Proc. Natl. Acad. Sci. U.S.A. 88:7170‐7174.
   Feil, R., Brocard, J., Mascrez, B., LeMeur, M., Metzger, D., and Chambon, P. 1996. Ligand‐activated site‐specific recombination in mice. Proc. Natl. Acad. Sci. U.S.A. 93:10887‐10890.
   Feil, R., Wagner, J., Metzger, D., and Chambon, P. 1997. Regulation of Cre recombinase activity by mutated estrogen receptor ligand‐binding domains. Biochem. Biophys. Res. Commun. 237:752‐757.
   Fiering, S., Kim, C.G., Epner, E.M., and Groudine, M. 1993. An “in‐out” strategy using gene targeting and FLP recombinase for the functional dissection of complex DNA regulatory elements: Analysis of the β‐globin locus control region. Proc. Natl. Acad. Sci. U.S.A. 90:8469‐8473.
   Fiering, S., Epner, E., Robinson, K., Zhuang, Y., Telling, A., Hu, M., Martin, D.I., Enver, T., Ley, T.J., and Groudine, M. 1995. Targeted deletion of 5′HS2 of the murine β‐globin LCR reveals that it is not essential for proper regulation of the β‐globin locus. Genes Dev. 9:2203‐2213.
   Fiering, S., Bender, M.A., and Groudine, M. 1999. Analysis of mammalian cis‐regulatory DNA elements by homolgous recombination. Methods Enzymol. 306:42‐66.
   Gu, H., Marth, J.D., Orban, P.C., Mossmann, H., and Rajewsky, K. 1994. Deletion of a DNA polymerase β gene segment in T cells using cell type–specific gene targeting. Science 265:103‐106.
   Hasty, P., Ramirez, S.R., Krumlauf, R., and Bradley, A. 1991. Introduction of a subtle mutation into the Hox‐2.6 locus in embryonic stem cells [published erratum appears in Nature, 1991, 353:94]. Nature 350:243‐246.
   Hunter, N.L., Awatramani, R.B., Farley, F.W., and Dymecki, S.M. 2005. Ligand‐activated Flpe for temporally regulated gene modifications. Genesis 41:99‐109.
   Kuhn, R., Schwenk, F., Aguet, M., and Rajewsky, K. 1995. Inducible gene targeting in mice. Science 269:1427‐1429.
   Mansour, S.L. 1990. Gene targeting in murine embryonic stem cells: Introduction of specific alterations into the mammalian genome. Genet. Anal. Tech. Appl. 7:219‐227.
   Mansour, S.L., Thomas, K.R., and Capecchi, M.R. 1988. Disruption of the proto‐oncogene int‐2 in mouse embryo‐derived stem cells: A general strategy for targeting mutations to nonselectable genes. Nature 336:348‐352.
   Mansour, S.L., Thomas, K.R., Deng, C.X., and Capecchi, M.R. 1990. Introduction of a lacZ reporter gene into the mouse int‐2 locus by homologous recombination. Proc. Natl. Acad. Sci. U.S.A. 87:7688‐7692.
   Mombaerts, P., Clarke, A.R., Hooper, M.L., and Tonegawa, S. 1991. Creation of a large genomic deletion at the T‐cell antigen receptor beta‐subunit locus in mouse embryonic stem cells by gene targeting. Proc. Natl. Acad. Sci. U.S.A. 88:3084‐3087.
   Mortensen, R.M., Zubiaur, M., Neer, E.J., and Seidman, J.G. 1991. Embryonic stem cells lacking a functional inhibitory G‐protein subunit (alpha i2) produced by gene targeting of both alleles. Proc. Natl. Acad. Sci. U.S.A. 88:7036‐7040.
   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.
   Olson, E.N., Arnold, H.H., Rigby, P.W., and Wold, B.J. 1996. Know your neighbors: Three phenotypes in null mutants of the myogenic bHLH gene MRF4. Cell 85:1‐4.
   Pham, C.T., MacIvor, D.M., Hug, B.A., Heusel, J.W., and Ley, T.J. 1996. Long‐range disruption of gene expression by a selectable marker cassette. Proc. Natl. Acad. Sci. U.S.A. 93:13090‐13095.
   Rajewsky, K., Gu, H., Kuhn, R., Betz, U.A., Muller, W., Roes, J., and Schwenk, F. 1996. Conditional gene targeting. J. Clin. Invest. 98:600‐603.
   Robertson, E.J. 1991. Using embryonic stem cells to introduce mutations into the mouse germ line. Biol. Reprod. 44:238‐245.
   Rodriguez, C.I., Buchholz, F., Galloway, J., Sequerra, R., Kasper, J., Ayala, R., Stewart, A.F., and Dymecki, S.M. 2000. High‐efficiency deleter mice show that FLPe is an alternative to Cre‐loxP. Nat. Genet. 25:139‐140.
   Rohlmann, A., Gotthardt, M., Willnow, T.E., Hammer, R.E., and Herz, J. 1996. Sustained somatic gene inactivation by viral transfer of Cre recombinase. Nat. Biotechnol. 14:1562‐1565.
   Sauer, B. 1993. Manipulation of transgenes by site‐specific recombination: Use of Cre recombinase. Methods Enzymol. 225:890‐900.
   Sauer, B. and Henderson, N. 1989. Cre‐stimulated recombination at loxP‐containing DNA sequences placed into the mammalian genome. Nucl. Acids Res. 17:147‐161.
   Schwenk, F., Baron, U., and Rajewsky, K. 1995. A cre‐transgenic mouse strain for the ubiquitous deletion of loxP‐flanked gene segments including deletion in germ cells. Nucl. Acids Res. 23:5080‐5081.
   Shibata, H., Toyama, K., Shioya, H., Ito, M., Hirota, M., Hasegawa, S., Matsumoto, H., Takano, H., Akiyama, T., Toyoshima, K., Kanamaru, R., Kanegae, Y., Saito, I., Nakamura, Y., Shiba, K., and Noda, T. 1997. Rapid colorectal adenoma formation initiated by conditional targeting of the Apc gene. Science 278:120‐123.
   St‐Onge, L., Furth, P.A., and Gruss, P. 1996. Temporal control of the Cre recombinase in transgenic mice by a tetracycline responsive promoter. Nucl. Acids Res. 24:3875‐3877.
   te Riele, H., Maandag, E.R., Clarke, A., Hooper, M., and Berns, A. 1990. Consecutive inactivation of both alleles of the pim‐1 proto‐oncogene by homologous recombination in embryonic stem cells. Nature 348:649‐651.
   van der Neut, R. 1997. Targeted gene disruption: Applications in neurobiology. J. Neurosci. Methods 71:19‐27.
   Vooijs, M., van der Valk, M., te Riele, H., and Berns, A. 1998. Flp‐mediated tissue‐specific inactivation of the retinoblastoma tumor suppressor gene in the mouse. Oncogene 17:1‐12.
   Wang, Y., Krushel, L.A., and Edelman, G.M. 1996. Targeted DNA recombination in vivo using an adenovirus carrying the cre recombinase gene. Proc. Natl. Acad. Sci. U.S.A. 93:3932‐3936.
   Williams‐Simons, L. and Westphal, H. 1999. EIIaCre: Utility of a general deleter strain. Transgenic Res. 8:53‐54.
   Yagi, T., Ikawa, Y., Yoshida, K., Shigetani, Y., Takeda, N., Mabuchi, I., Yamamoto, T., and Aizawa, S. 1990. Homologous recombination at c‐fyn locus of mouse embryonic stem cells with use of diphtheria toxin A‐fragment gene in negative selection. Proc. Natl. Acad. Sci. U.S.A. 87:9918‐9922.
   Zhang, Y., Riesterer, C., Ayrall, A.M., Sablitzky, F., Littlewood, T.D., and Reth, M. 1996. Inducible site‐directed recombination in mouse embryonic stem cells. Nucl. Acids Res. 24:543‐548.
   Zimmer, A. 1992. Manipulating the genome by homologous recombination in embryonic stem cells. Annu. Rev. Neurosci. 15:115‐137.
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