Abstract
One of the most effective approaches for determining gene function involves engineering mice with mutations or deletions in endogenous genes of interest. Historically, this approach has been limited by the difficulty and time required to generate such mice. We describe the development of a high-throughput and largely automated process, termed VelociGene, that uses targeting vectors based on bacterial artificial chromosomes (BACs). VelociGene permits genetic alteration with nucleotide precision, is not limited by the size of desired deletions, does not depend on isogenicity or on positive–negative selection, and can precisely replace the gene of interest with a reporter that allows for high-resolution localization of target-gene expression. We describe custom genetic alterations for hundreds of genes, corresponding to about 0.5–1.0% of the entire genome. We also provide dozens of informative expression patterns involving cells in the nervous system, immune system, vasculature, skeleton, fat and other tissues.
*Note: In the author list of the AOP version of this article, the name of author Rostislav Chernomorsky was misspelled Rostislav Chernomorski. This has been corrected in the online and print versions of the article.
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Change history
30 May 2003
replaced PDF with correct version (print was correct), ammended PDF with errata, footnote in SGML at abstract
References
Lander, E.S. et al. Initial sequencing and analysis of the human genome. Nature 409, 860–921 (2001).
Venter, J.C. et al. The sequence of the human genome. Science 291, 1304–1351 (2001).
Goldstein, J.L. Laskers for 2001: knockout mice and test-tube babies. Nat. Med. 7, 1079–1080 (2001).
Evans, M.J. The cultural mouse. Nat. Med. 7, 1081–1083 (2001).
Capecchi, M.R. Generating mice with targeted mutations. Nat. Med. 7, 1086–1090 (2001).
Smithies, O. Forty years with homologous recombination. Nat. Med. 7, 1083–1086 (2001).
Cheah, S.S. & Behringer, R.R. Contemporary gene targeting strategies for the novice. Mol. Biotechnol. 19, 297–304 (2001).
Hrabe de Angelis, M.H. et al. Genome-wide, large-scale production of mutant mice by ENU mutagenesis. Nat. Genet. 25, 444–447 (2000).
Nolan, P.M. et al. A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse. Nat. Genet. 25, 440–443 (2000).
Zambrowicz, B.P. et al. Disruption and sequence identification of 2,000 genes in mouse embryonic stem cells. Nature 392, 608–611 (1998).
Mitchell, K.J. et al. Functional analysis of secreted and transmembrane proteins critical to mouse development. Nat. Genet. 28, 241–249 (2001).
Leighton, P.A. et al. Defining brain wiring patterns and mechanisms through gene trapping in mice. Nature 410, 174–179 (2001).
Yang, X.W., Model, P. & Heintz, N. Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat. Biotechnol. 15, 859–865 (1997).
Yang, X.W., Wynder, C., Doughty, M.L. & Heintz, N. BAC-mediated gene-dosage analysis reveals a role for Zipro1 (Ru49/Zfp38) in progenitor cell proliferation in cerebellum and skin. Nat. Genet. 22, 327–335 (1999).
Lee, E.C. et al. A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73, 56–65 (2001).
Heintz, N. BAC to the future: the use of bac transgenic mice for neuroscience research. Nat. Rev. Neurosci. 2, 861–870 (2001).
Joyner, A.L. (ed.) Gene Targeting; a Practical Approach, edn. 2 (Oxford University Press, New York, 1999).
Hasty, P., Rivera-Perez, J. & Bradley, A. The length of homology required for gene targeting in embryonic stem cells. Mol. Cell Biol. 11, 5586–5591 (1991).
Deng, C. & Capecchi, M.R. 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 (1992).
Zhang, H., Hasty, P. & Bradley, A. Targeting frequency for deletion vectors in embryonic stem cells. Mol. Cell Biol. 14, 2404–2410 (1994).
Waldman, A.S. & Liskay, R.M. Dependence of intrachromosomal recombination in mammalian cells on uninterrupted homology. Mol. Cell Biol. 8, 5350–5357 (1988).
te Riele, H., Maandag, E.R. & Berns, A. Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs. Proc. Natl. Acad. Sci. USA 89, 5128–5132 (1992).
Mansour, S.L., Thomas, K.R. & Capecchi, M.R. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336, 348–352 (1988).
Mansour, S.L., Thomas, K.R., Deng, C.X. & Capecchi, M.R. Introduction of a lacZ reporter gene into the mouse int-2 locus by homologous recombination. Proc. Natl. Acad. Sci. USA 87, 7688–7692 (1990).
Puri, M.C., Rossant, J., Alitalo, K., Bernstein, A. & Partanen, J. The receptor tyrosine kinase TIE is required for integrity and survival of vascular endothelial cells. EMBO J. 14, 5884–5891 (1995).
Miquerol, L., Gertsenstein, M., Harpal, K., Rossant, J. & Nagy, A. Multiple developmental roles of VEGF suggested by a lacZ-tagged allele. Dev. Biol. 212, 307–322 (1999).
Zhang, Y., Buchholz, F., Muyrers, J.P. & Stewart, A.F. A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 20, 123–128 (1998).
Muyrers, J.P., Zhang, Y., Testa, G. & Stewart, A.F. Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res. 27, 1555–1557 (1999).
Narayanan, K., Williamson, R., Zhang, Y., Stewart, A.F. & Ioannou, P.A. Efficient and precise engineering of a 200 kb β-globin human/bacterial artificial chromosome in E. coli DH10B using an inducible homologous recombination system. Gene Ther. 6, 442–447 (1999).
Angrand, P.O., Daigle, N., van der Hoeven, F., Scholer, H.R. & Stewart, A.F. Simplified generation of targeting constructs using ET recombination. Nucleic Acids Res. 27, e16 (1999).
Yu, D. et al. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. USA 97, 5978–5983 (2000).
Muyrers, J.P., Zhang, Y. & Stewart, A.F. Techniques: recombinogenic engineering–new options for cloning and manipulating DNA. Trends Biochem. Sci. 26, 325–331 (2001).
Gregory, S.G. et al. A physical map of the mouse genome. Nature 418, 743–750 (2002).
Bodine, S.C. et al. Identification of ubiquitin ligases required for skeletal muscle atrophy. Science 294, 1704–1708 (2001).
Testa, G. et al. Large BAC-based targeting constructs present new options for genomic engineering. Nat. Biotechnol. 21, 443–447 (2003).
Matise, M., Auerbach, W. & Joyner, A.L. in Gene Targeting: A Practical Approach (ed. Joyner, A.L.) 101–132 (Oxford University Press, New York, 2000).
Swiatek, P.J. & Gridley, T. Perinatal lethality and defects in hindbrain development in mice homozygous for a targeted mutation of the zinc finger gene Krox20. Genes Dev. 7, 2071–2084 (1993).
Auerbach, W. et al. Establishment and chimera analysis of 129/SvEv- and C57BL/6-derived mouse embryonic stem cell lines. Biotechniques 29, 1024–1028, 1030, 1032 (2000).
Schuster-Gossler, K. et al. Use of coisogenic host blastocysts for efficient establishment of germline chimeras with C57BL/6J ES cell lines. Biotechniques 31, 1022–1024, 1026 (2001).
Lundsteen, C. & Lind, A.M. A test of a climate room for preparation of chromosome slides. Clin. Genet. 28, 260–262 (1985).
Pinkel, D., Straume, T. & Gray, J.W. Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc. Natl. Acad. Sci. USA 83, 2934–2938 (1986).
Lichter, P., Cremer, T., Borden, J., Manuelidis, L. & Ward, D.C. Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum. Genet. 80, 224–234 (1988).
Pecker, I. et al. Identification and chromosomal localization of Atm, the mouse homolog of the ataxia-telangiectasia gene. Genomics 35, 39–45 (1996).
Suri, C. et al. Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 87, 1171–1180 (1996).
Acknowledgements
We thank Margaret Karow for intellectual input, Petra Kraus for analysis of gene expression patterns and Fred W. Alt, Craig Bassing, Richard Flavell, Mike Brown, Joe Goldstein, Eric Shooter and Kornelia Polyak for suggesting genes to target. We thank Amalia Dutra for advice and assistance with FISH. We would also like to thank Li Pan, Joyce McClain, Virginia Hughes, Jeffrey Vercollone, Kethi Mullei, Jorge Bermudez, Jennifer Leung, Ning Yuan, Samantha Park, Ruth Santos, Collen Correa, Huan Jiang, Jinsop Om, Youngli Chang, Haige Zhang and Jose Rodriguez for technical assistance, as well as Vicki Lan and Scott Staton for graphics support.
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The authors of this paper (except for A.F.S.) are employed at Regeneron Pharmaceuticals, Inc.
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Valenzuela, D., Murphy, A., Frendewey, D. et al. High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat Biotechnol 21, 652–659 (2003). https://doi.org/10.1038/nbt822
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DOI: https://doi.org/10.1038/nbt822
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