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. 1993 May 11;21(9):2025–2029. doi: 10.1093/nar/21.9.2025

Genomic targeting with purified Cre recombinase.

W Baubonis 1, B Sauer 1
PMCID: PMC309460  PMID: 8502542

Abstract

Purified Cre recombinase protein introduced directly into cultured mammalian cells by lipofection catalyzes both site-specific chromosomal integration of a co-transfected lox targeting vector and precise excision of genomic DNA flanked by directly repeated lox sites. This procedure eliminates the need to transfect cre expression plasmids to activate recombination at lox sites. We used this simplified procedure to investigate the effect on targeting efficiency of both lox vector design and chromosomal position of the lox target. We show that such chromosomal position effects can exert at least a 50-fold per lox target difference in targeting efficiency in a human osteosarcoma cell line.

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Selected References

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  1. Fukushige S., Sauer B. Genomic targeting with a positive-selection lox integration vector allows highly reproducible gene expression in mammalian cells. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):7905–7909. doi: 10.1073/pnas.89.17.7905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Golic K. G., Lindquist S. The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell. 1989 Nov 3;59(3):499–509. doi: 10.1016/0092-8674(89)90033-0. [DOI] [PubMed] [Google Scholar]
  3. Kashanchi F., Duvall J. F., Brady J. N. Electroporation of viral transactivator proteins into lymphocyte suspension cells. Nucleic Acids Res. 1992 Sep 11;20(17):4673–4674. doi: 10.1093/nar/20.17.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Lakso M., Sauer B., Mosinger B., Jr, Lee E. J., Manning R. W., Yu S. H., Mulder K. L., Westphal H. Targeted oncogene activation by site-specific recombination in transgenic mice. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6232–6236. doi: 10.1073/pnas.89.14.6232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Maeser S., Kahmann R. The Gin recombinase of phage Mu can catalyse site-specific recombination in plant protoplasts. Mol Gen Genet. 1991 Nov;230(1-2):170–176. doi: 10.1007/BF00290665. [DOI] [PubMed] [Google Scholar]
  6. Nakamura N., Sugino H., Takahara K., Jin C., Fukushige S., Matsubara K. Endogenous retroviral LTR DNA sequences as markers for individual human chromosomes. Cytogenet Cell Genet. 1991;57(1):18–22. doi: 10.1159/000133105. [DOI] [PubMed] [Google Scholar]
  7. Nickoloff J. A. Transcription enhances intrachromosomal homologous recombination in mammalian cells. Mol Cell Biol. 1992 Dec;12(12):5311–5318. doi: 10.1128/mcb.12.12.5311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. O'Gorman S., Fox D. T., Wahl G. M. Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science. 1991 Mar 15;251(4999):1351–1355. doi: 10.1126/science.1900642. [DOI] [PubMed] [Google Scholar]
  9. Onouchi H., Yokoi K., Machida C., Matsuzaki H., Oshima Y., Matsuoka K., Nakamura K., Machida Y. Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells. Nucleic Acids Res. 1991 Dec 11;19(23):6373–6378. doi: 10.1093/nar/19.23.6373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Orban P. C., Chui D., Marth J. D. Tissue- and site-specific DNA recombination in transgenic mice. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6861–6865. doi: 10.1073/pnas.89.15.6861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Pepperkok R., Zanetti M., King R., Delia D., Ansorge W., Philipson L., Schneider C. Automatic microinjection system facilitates detection of growth inhibitory mRNA. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6748–6752. doi: 10.1073/pnas.85.18.6748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Sanes J. R., Rubenstein J. L., Nicolas J. F. Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos. EMBO J. 1986 Dec 1;5(12):3133–3142. doi: 10.1002/j.1460-2075.1986.tb04620.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Sauer B. Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1987 Jun;7(6):2087–2096. doi: 10.1128/mcb.7.6.2087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Sauer B., Henderson N. Cre-stimulated recombination at loxP-containing DNA sequences placed into the mammalian genome. Nucleic Acids Res. 1989 Jan 11;17(1):147–161. doi: 10.1093/nar/17.1.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Sauer B., Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5166–5170. doi: 10.1073/pnas.85.14.5166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sauer B., Henderson N. Targeted insertion of exogenous DNA into the eukaryotic genome by the Cre recombinase. New Biol. 1990 May;2(5):441–449. [PubMed] [Google Scholar]
  17. Sauer B., Whealy M., Robbins A., Enquist L. Site-specific insertion of DNA into a pseudorabies virus vector. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9108–9112. doi: 10.1073/pnas.84.24.9108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Wigler M., Levy D., Perucho M. The somatic replication of DNA methylation. Cell. 1981 Apr;24(1):33–40. doi: 10.1016/0092-8674(81)90498-0. [DOI] [PubMed] [Google Scholar]
  19. Winegar R. A., Phillips J. W., Youngblom J. H., Morgan W. F. Cell electroporation is a highly efficient method for introducing restriction endonucleases into cells. Mutat Res. 1989 Jan-Feb;225(1-2):49–53. doi: 10.1016/0165-7992(89)90032-8. [DOI] [PubMed] [Google Scholar]
  20. Wolff J. A., Ludtke J. J., Acsadi G., Williams P., Jani A. Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle. Hum Mol Genet. 1992 Sep;1(6):363–369. doi: 10.1093/hmg/1.6.363. [DOI] [PubMed] [Google Scholar]

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