Simultaneous Cre catalyzed recombination of two alleles to restore neomycin sensitivity and facilitate homozygous mutations.
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.
Milstone
D S
DS
Vascular Research Division, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Bradwin
G
G
Mortensen
R M
RM
D000483:Alleles; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002460:Cell Line; D043525:Cytosine Deaminase; D004351:Drug Resistance / Q000235:genetics; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019204:GTP-Binding Proteins / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005819:Genetic Markers; D005839:Gentamicins / Q000494:pharmacology; D006720:Homozygote; D019426:Integrases / Q000378:metabolism*; D019656:Loss of Heterozygosity; D051379:Mice; D009154:Mutation; D009355:Neomycin / Q000494:pharmacology*; D009700:Nucleoside Deaminases / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics; D011995:Recombination, Genetic; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics; D014764:Viral Proteins
D000900:Anti-Bacterial Agents; D005819:Genetic Markers; D005839:Gentamicins; D011518:Proto-Oncogene Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D014764:Viral Proteins; C010680:antibiotic G 418; D013937:Thymidine Kinase; C045073:Cre recombinase; D019426:Integrases; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase; D019204:GTP-Binding Proteins; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C498016:Gnai2 protein, mouse; D009355:Neomycin