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. 1999 Aug 1;27(15):e10. doi: 10.1093/nar/27.15.e10

Simultaneous Cre catalyzed recombination of two alleles to restore neomycin sensitivity and facilitate homozygous mutations.

D S Milstone 1, G Bradwin 1, R M Mortensen 1
PMCID: PMC148521  PMID: 10454629

Abstract

Cells homozygous for neo-expressing mutations can be derived by culturing heterozygotes with elevated G418. We demonstrate that this strategy is significantly less efficient if hyg is substituted for neo. Therefore, to introduce additional mutations Cre recombinase was used to remove floxed neo from both alleles of homozygotes at two different loci. The rate-determining step in Cre excision appeared independent of substrate copy number. Incorporating cytosine deaminase and Herpes simplex virus thymidine kinase allowed negative selection for both targeting and Cre excision. The resulting G418-sensitive homozygous mutants should allow mutagenesis at additional loci and avoid untoward effects of retained selection markers.

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Selected References

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  1. Abuin A., Bradley A. Recycling selectable markers in mouse embryonic stem cells. Mol Cell Biol. 1996 Apr;16(4):1851–1856. doi: 10.1128/mcb.16.4.1851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bories J. C., Demengeot J., Davidson L., Alt F. W. Gene-targeted deletion and replacement mutations of the T-cell receptor beta-chain enhancer: the role of enhancer elements in controlling V(D)J recombination accessibility. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7871–7876. doi: 10.1073/pnas.93.15.7871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Gu H., Marth J. D., Orban P. C., Mossmann H., Rajewsky K. Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science. 1994 Jul 1;265(5168):103–106. doi: 10.1126/science.8016642. [DOI] [PubMed] [Google Scholar]
  4. Kühn R., Schwenk F., Aguet M., Rajewsky K. Inducible gene targeting in mice. Science. 1995 Sep 8;269(5229):1427–1429. doi: 10.1126/science.7660125. [DOI] [PubMed] [Google Scholar]
  5. 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. 1988 Nov 24;336(6197):348–352. doi: 10.1038/336348a0. [DOI] [PubMed] [Google Scholar]
  6. Metzger J. M., Lin W. I., Samuelson L. C. Transition in cardiac contractile sensitivity to calcium during the in vitro differentiation of mouse embryonic stem cells. J Cell Biol. 1994 Aug;126(3):701–711. doi: 10.1083/jcb.126.3.701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Mortensen R. M., Conner D. A., Chao S., Geisterfer-Lowrance A. A., Seidman J. G. Production of homozygous mutant ES cells with a single targeting construct. Mol Cell Biol. 1992 May;12(5):2391–2395. doi: 10.1128/mcb.12.5.2391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Mortensen R. M., Seidman J. G. Inactivation of G-protein genes: double knockout in cell lines. Methods Enzymol. 1994;237:356–366. doi: 10.1016/s0076-6879(94)37075-3. [DOI] [PubMed] [Google Scholar]
  9. Mortensen R. M., Zubiaur M., Neer E. J., Seidman J. G. 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. 1991 Aug 15;88(16):7036–7040. doi: 10.1073/pnas.88.16.7036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Mullen C. A., Kilstrup M., Blaese R. M. Transfer of the bacterial gene for cytosine deaminase to mammalian cells confers lethal sensitivity to 5-fluorocytosine: a negative selection system. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):33–37. doi: 10.1073/pnas.89.1.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Olson E. N., Arnold H. H., Rigby P. W., Wold B. J. Know your neighbors: three phenotypes in null mutants of the myogenic bHLH gene MRF4. Cell. 1996 Apr 5;85(1):1–4. doi: 10.1016/s0092-8674(00)81073-9. [DOI] [PubMed] [Google Scholar]
  12. Pham C. T., MacIvor D. M., Hug B. A., Heusel J. W., Ley T. J. Long-range disruption of gene expression by a selectable marker cassette. Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):13090–13095. doi: 10.1073/pnas.93.23.13090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Sauer B. Recycling selectable markers in yeast. Biotechniques. 1994 Jun;16(6):1086–1088. [PubMed] [Google Scholar]
  15. Sowell M. O., Ye C., Ricupero D. A., Hansen S., Quinn S. J., Vassilev P. M., Mortensen R. M. Targeted inactivation of alphai2 or alphai3 disrupts activation of the cardiac muscarinic K+ channel, IK+Ach, in intact cells. Proc Natl Acad Sci U S A. 1997 Jul 22;94(15):7921–7926. doi: 10.1073/pnas.94.15.7921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Yenofsky R. L., Fine M., Pellow J. W. A mutant neomycin phosphotransferase II gene reduces the resistance of transformants to antibiotic selection pressure. Proc Natl Acad Sci U S A. 1990 May;87(9):3435–3439. doi: 10.1073/pnas.87.9.3435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Zhu Y., Qi C., Korenberg J. R., Chen X. N., Noya D., Rao M. S., Reddy J. K. Structural organization of mouse peroxisome proliferator-activated receptor gamma (mPPAR gamma) gene: alternative promoter use and different splicing yield two mPPAR gamma isoforms. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7921–7925. doi: 10.1073/pnas.92.17.7921. [DOI] [PMC free article] [PubMed] [Google Scholar]

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