Targeting vector design and confirmation of targeting
John M. Hinz, Tebbs, Robert S., Wilson, Paul F., Nham, Peter B., Salazar, Edmund P., Hatsumi Nagasawa, Salustra S. Urbin, Bedford, Joel S., Thompson, Larry H. · Figshare · 2011
Copyright information:Taken from "Repression of mutagenesis by Rad51D-mediated homologous recombination"Nucleic Acids Research 2006;34(5):1358-1368.Published online 6 Mar 2006PMCID:PMC1390685.© The Author 2006. Published by Oxford University Press. All rights reserved () Configuration of targeting vectors. The targeting vectors contained a gene for negative selection and, for positive selection, a gene for one allele and a gene for the other allele. Step 1 results in a targeted recombination event in which exon 4 is replaced by a functional exon 4 flanked by / and LoxP sites. After each allele has been targeted, without affecting function, treatment with transfected Cre recombinase in step 2 causes deletion of exon 4 and loss of expression of functional Rad51D. () Screening for exon 4 deletion events in both alleles. After Cre transfection and phenotypic expression, single-cell clones were picked, distributed into 24-well trays, grown for 8 days in media containing, geneticin, puromycin, MMC or no drug, and then stained. The picture shows a tray in which four of five clones had acquired sensitivity to each selection agent. Some clones (data not shown) that were sensitive to only to geneticin or puromycin had presumably experienced recombination in one allele only. () Western blot of Rad51D protein in parental, mutant and gene-complemented mutant cells. () DNA profiles of mutant and control cell lines.