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Nucleic Acids Res. 1999 Aug 1;27(15):e10.

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

Author information

1
Vascular Research Division, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

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.

PMID:
10454629
PMCID:
PMC148521
DOI:
10.1093/nar/27.15.e10
[Indexed for MEDLINE]
Free PMC Article

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