Abstract
Embryonic stem (ES) cell-mediated transgenic approaches have revolutionized mammalian genetics in the last decade. Close to 4000 genes have been targeted to date. Analysis of these genetic alterations has provided an unprecedented understanding of critical gene functions that underlie normal developmental and disease mechanisms in mammals. We have also learned, however, that mammalian genetic determination is complex: genes have multiple functions during the life of an individual. In addition, mammalian genetic determination often utilizes gene families, in which the members have similar structure and overlapping expression and functions. These two phenomena pointed out some limitations of the gene-targeting approach. In many cases a gene “knockout” resulted in early embryonic lethality, which obscured the study of potential later functions. In other cases the “knockout” did not have any phenotype due to the compensation of the gene deficiency by other family members. These limitations have called for further development of the powerful gene-targeting technology (1–3).
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References
Nagy, A. and Rossant, J. (1996) Targeted mutagenesis: analysis of phenotype without germ line transmission. J. Clin. Invest. 97, 1360–1365.
Rossant, J. and Nagy A. (1995) Genome engineering: the new mouse genetics. Nat. Med. 1, 592–594.
Lobe, C. and Nagy, A. (1997) Conditional genome alterations. BioEssay 20, 200–208.
Kilby, N. J., Snaith, M. R., and Murray, J. A. H. (1993) Site-specific recombinases: tools for genome engineering. Trends Genet. 9, 413–421.
Dymecki, S. M. (1996) Flp recombinase promotes site-specific DNA recombination in embryonic stem cells and transgenic mice. Proc. Natl. Acad. Sci. USA 93, 6191–6196.
Sauer, B. and Henderson, N. (1988) Site-specific DNA recombination in mamalian cells by the Cre recombinase of bacteriophage P1. Proc. Natl. Acad. Sci. USA 85, 5166–5270.
Argos, P., Landy, A., Abremski, K., et al. (1986) The integrase family of site-specific recombinases: regional similarities and global diversity. EMBO J. 5, 433–440.
Hoess, R. H. and Abremski, K. (1985) Mechanism of strand cleavage and exchange in the Cre-lox site-specific recombination system. J. Mol. Biol. 181, 351–362.
Sauer, B. and Henderson, N. (1989) Cre-stimulated recombination at loxP-containing DNA equences placed into the mammalian genome. Nucleic Acid Res. 17, 147–161.
Sauer, B. and Henderson, N. (1990) Targeted insertion of exogenous DNA into the eukaryotic genome by the Cre recombinase. New Biologist 2, 441–449.
Lakso, M., Sauer, B., B. Mosinger, J., et al. (1992) Targeted oncogene activity by site-specific recombination in transgenic mice. Proc. Natl. Acad. Sci. USA 89, 6232–6236.
Gu, H., Zou, Y. R., and Rajewsky, K. (1993) Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell 73, 1155–1164.
Gordon, J. W., Scangos, G. A., Plotkin, D. J., et al. (1980) Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl. Acad. Sci. USA 77, 7380–7384.
Brinster, R. L., Chen, H. Y., and Trumbauer, M. E. (1981) Mouse oocytes transcribe injected Xenopus 5S RNA gene. Science 211, 396–398.
Brinster, R. L., Chen, H. Y., Trumbauer, M., Senear, A. W., et al. (1981) Somatic expression of herpes thymidine kinase in mice following injection of fusion gene into eggs. Cell 27, 223–231.
Palmiter, R. D. and Brinster, R. L. (1986) Germ-line transformation of mice. Annu. Rev. Genet. 20, 645–499.
Lewandoski, M. and Martin, G. R. (1997) Cre-mediated chromosome loss in mice. Nat. Genet. 17, 223–225.
Meyers, E. N., Lewandoski, M., Martin, G. R. (1998) An Fgf8 mutant allelic series generated by Cre-and Flp-mediated recombination. Nat. Genet. 18, 136–141.
Gu, H., Marth, J. D., Orban, P. C., et al. (1994) Deletion of a DNA polymerase β gene segment in T cells using cell type-specific gene targeting. Science 265, 103–106.
Tsien, J. Z., Huerta, P. T., Tonegawa, S. (1996) The essential role of hippocampal CA1 NMDA receptor-dependent synaptic plasticity in spatial memory. Cell 87, 1327–1338.
Nagy, A., Moens, C. B., Ivanyi, E., et al. (1998) Dissecting the role of N-myc in development using a single targeting vector to generate a series of alleles. Curr. Biol. 8, 661–664.
Jacks, T., Shih, T. S., Schmitt, E. M., et al. (1994) Tumor predisposition in mice heterozygous for a targeted mutation in Nf1. Nat. Genet. 7, 353–361.
Carmeliet, P., Ferreira, V., Breier, G., et al. (1996) Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380, 435–439.
Moens, C. B., Auerbach, A. B., Conlon, R. A., et al. (1992) A targeted mutation reveals a role for N-myc in branching morphogenesis in the embryonic mouse lung. Genes Dev. 6, 691–704.
St.-Onge, L., Furth, P. A., and Gruss, P. (1996) Temporal control of the Cre recombinase in transgenic mice by tetracycline responsive promoter. Nucleic Acids Res. 24, 3875–3877.
Brocard, J., Warot, X., Wendling, O., et al. (1997) Spatio-temporally controlled site-specific somatic mutagenesis in the mouse. Proc. Natl. Acad. Sci. USA 94, 14,559–14,563.
Schwenk, F., Kuhn, R., Angrand, P. O., et al. (1998) Temporally and spatially regulated somatic mutagenesis in mice. Nucleic Acid Res. 26, 1427–1432.
Sakai, K. and Miyazaki, J. (1997) A transgenic mouse line that retains Cre recombinase activity in mature oocytes irrespective of the Cre transgene transmission. Biochem. Biophys. Res. Commun. 237, 318–324.
Araki, K., Araki, M., Miyazaki, J., and Vassalli, P. (1995) Site-specific recombination of a transgene in fertilized eggs by transient expression of Cre recombinase. Proc. Natl. Acad. Sci. USA 92, 160–164.
Ramirez-Slois, R., Liu, P., and Bradley, A. (1995) Chromosome engineering in mice. Nature 378, 720–724.
Smith, A. J. H., Sousa, M. A. D., Kwabi-Addo, et al. (1995) A site-directed chromosomal translocation induced in embryonic stem cells by Cre-loxP recombination. Nat. Genet. 9, 376–385.
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© 2001 Humana Press Inc., Totowa, NJ
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Nagy, A., Mar, L. (2001). Creation and Use of a Cre Recombinase Transgenic Database. In: Tymms, M.J., Kola, I. (eds) Gene Knockout Protocols. Methods in Molecular Biology, vol 158. Humana Press. https://doi.org/10.1385/1-59259-220-1:95
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DOI: https://doi.org/10.1385/1-59259-220-1:95
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