Design and Generation of Gene‐Targeting Vectors

Benedikt Wefers1, Wolfgang Wurst2, Ralf Kühn3

1 German Research Center for Environmental Health, Munich, Germany, 2 Deutsches Zentrum für Neurodegenerative Erkrankungen e.V. (DZNE), Munich, Germany, 3 Technical University Munich, Munich, Germany
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo100179
Online Posting Date:  March, 2011
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


This unit provides an overview of the major types of mutant alleles that can be generated by gene targeting in ES cells. It presents the growing public resources of premade gene targeting vectors, modified ES cells, and mutant mice. General guidelines for the design of targeting vectors are followed by protocols for the construction of vectors to generate knockout (KO), conditional KO, and subtle mutant alleles. Curr. Protoc. Mouse Biol. 1:199‐211. © 2011 by John Wiley & Sons, Inc.

Keywords: gene targeting; conditional KO mice; targeting vector design; knockout mice; EUCOMM/KOMP

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Protocols for the Construction of Gene Targeting Vectors
  • Concluding Remarks
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Branda, C.S. and Dymecki, S.M. 2004. Talking about a revolution: The impact of site‐specific recombinases on genetic analyses in mice. Dev. Cell 6:7‐28.
   Capecchi, M.R. 1989. The new mouse genetics: Altering the genome by gene targeting. Trends Genet. 5:70‐76.
   Capecchi, M.R. 2005. Gene targeting in mice: Functional analysis of the mammalian genome for the twenty‐first century. Nat. Rev. Genet. 6:507‐512.
   Chan, W., Costantino, N., Li, R., Lee, S.C., Su, Q., Melvin, D., Court, D.L., and Liu, P. 2007. A recombineering based approach for high‐throughput conditional knockout targeting vector construction. Nucleic Acids Res. 35:e64.
   Cormack, B. 2001. Directed mutagenesis using the polymerase chain reaction.Curr. Protoc. Mol. Biol. 37:8.5.1‐8.5.10.
   Eggan, K., Akutsu, H., Loring, J., Jackson‐Grusby, L., Klemm, M., Rideout, W.M. 3rd, Yanagimachi, R., and Jaenisch, R. 2001. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc. Natl. Acad. Sci. U.S.A. 98:6209‐6214.
   Elion, E.A., Marina, P., and Yu, L. 2007. Constructing recombinant DNA molecules by PCR. Curr. Protoc. Mol. Biol. 78:3.17.1‐3.17.12.
   Evans, M.J., Carlton, M.B., and Russ, A.P. 1997. Gene trapping and functional genomics. Trends Genet. 13:370‐374.
   Friedel, R.H., Seisenberger, C., Kaloff, C., and Wurst, W. 2007. EUCOMM–the European conditional mouse mutagenesis program. Brief. Funct. Genomic Proteomic 6:180‐185.
   Fu, J., Teucher, M., Anastassiadis, K., Skarnes, W., and Stewart, A.F. 2010. A recombineering pipeline to make conditional targeting constructs. Methods Enzymol. 477:125‐144.
   Gomez‐Rodriguez, J., Washington, V., Cheng, J., Dutra, A., Pak, E., Liu, P., McVicar, D.W., and Schwartzberg, P.L. 2008. Advantages of q‐PCR as a method of screening for gene targeting in mammalian cells using conventional and whole BAC‐based constructs. Nucleic Acids Res. 36:e117.
   Hansen, J., Floss, T., Van Sloun, P., Fuchtbauer, E.M., Vauti, F., Arnold, H.H., Schnutgen, F., Wurst, W., von Melchner, H., and Ruiz, P. 2003. A large‐scale, gene‐driven mutagenesis approach for the functional analysis of the mouse genome. Proc. Natl. Acad. Sci. U.S.A. 100:9918‐9922.
   Hasty, P., Abuin, A., and Bradley, A. 2000. Gene targeting, principles, and practice in mammalian cells. In Gene Targeting: A Practical Approach, 2nd ed. (A.L. Joyner, ed.) pp. 1‐35. Oxford University Press, Oxford.
   Hitz, C., Steuber‐Buchberger, P., Delic, S., Wurst, W., and Kuhn, R. 2009. Generation of shRNA transgenic mice. Methods Mol. Biol. 530:101‐129.
   Kranz, A., Fu, J., Duerschke, K., Weidlich, S., Naumann, R., Stewart, A.F., and Anastassiadis, K. 2010. An improved Flp deleter mouse in C57Bl/6 based on Flpo recombinase. Genesis 48:512‐520.
   Kühn, R. and Wurst, W. 2005. Mouse mutagenesis and gene function. In Encyclopedia of Genetics, Genomics, Proteomics and Bioinformatics, p. 18. John Wiley & Sons, Hoboken, N.J.
   Kwan, K.M. 2002. Conditional alleles in mice: Practical considerations for tissue‐specific knockouts. Genesis 32:49‐62.
   Lee, S.C., Wang, W., and Liu, P. 2009. Construction of gene‐targeting vectors by recombineering. Methods Mol. Biol. 530:15‐27.
   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.
   Nagy, A., Gertsenstein, M., Vintersten, K., and Behringer, R. 2003. Manipulating the Mouse Embryo, third edition ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
   Pettitt, S.J., Liang, Q., Rairdan, X.Y., Moran, J.L., Prosser, H.M., Beier, D.R., Lloyd, K.C., Bradley, A., and Skarnes, W.C. 2009. Agouti C57BL/6N embryonic stem cells for mouse genetic resources. Nat. Methods 6:493‐495.
   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.
   Schnutgen, F., Doerflinger, N., Calleja, C., Wendling, O., Chambon, P., and Ghyselinck, N.B. 2003. A directional strategy for monitoring Cre‐mediated recombination at the cellular level in the mouse. Nat. Biotechnol. 21:562‐565.
   Schnutgen, F., De‐Zolt, S., Van Sloun, P., Hollatz, M., Floss, T., Hansen, J., Altschmied, J., Seisenberger, C., Ghyselinck, N.B., Ruiz, P., Chambon, P., Wurst, W., and von Melchner, H. 2005. Genomewide production of multipurpose alleles for the functional analysis of the mouse genome. Proc. Natl. Acad. Sci. U.S.A. 102:7221‐7226.
   Southern, E. 2006. Southern blotting. Nat. Protoc. 1:518‐525.
   te Riele, 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.
   Testa, G., Zhang, Y., Vintersten, K., Benes, V., Pijnappel, W.W., Chambers, I., Smith, A.J., Smith, A.G., and Stewart, A.F. 2003. Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles. Nat. Biotechnol. 21:443‐447.
   Testa, G., Vintersten, K., Zhang, Y., Benes, V., Muyrers, J.P., and Stewart, A.F. 2004. BAC engineering for the generation of ES cell‐targeting constructs and mouse transgenes. Methods Mol. Biol. 256:123‐139.
   Thomas, K.R. and Capecchi, M.R. 1987. Site‐directed mutagenesis by gene targeting in mouse embryo‐derived stem cells. Cell 51:503‐512.
   Torres, R.M. and Kühn, R. 1997. Laboratory Protocols for Conditional Gene Targeting. Oxford University Press, Oxford.
   Valenzuela, D.M., Murphy, A.J., Frendewey, D., Gale, N.W., Economides, A.N., Auerbach, W., Poueymirou, W.T., Adams, N.C., Rojas, J., Yasenchak, J., Chernomorsky, R., Boucher, M., Elsasser, A.L., Esau, L., Zheng, J., Griffiths, J.A., Wang, X., Su, H., Xue, Y., Dominguez, M.G., Noguera, I., Torres, R., Macdonald, L.E., Stewart, A.F., DeChiara, T.M., and Yancopoulos, G.D. 2003. High‐throughput engineering of the mouse genome coupled with high‐resolution expression analysis. Nat. Biotechnol. 21:652‐659.
   Wu, S., Ying, G., Wu, Q., and Capecchi, M.R. 2008. A protocol for constructing gene targeting vectors: Generating knockout mice for the cadherin family and beyond. Nat. Protoc. 3:1056‐1076.
   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.
PDF or HTML at Wiley Online Library