Pot1a^tm1.2Blas

An arginine to cysteine substitution at amino acid 117 was knocked-in using the FLEx switch system. A cassette containing the following elements replaced the wild-type exon 7: a loxP site, a loxP511 site, exon 7 with a mutation changing arginine codon 117 (CGC) to cysteine (TGC) (p.R117C) in reverse orientation, an FRT site flanked neomycin selection cassette, a loxP site in reverse orientation, wild-type exon 7 in forward orientation, and a loxP511 site in reverse orientation. The neomycin selection cassette was removed via Flp-mediated recombination. Cre-mediated recombination removed the wild-type exon 7 and replaced it with the mutated exon, harboring the R117C missense mutation, in forward orientation. Western blot analysis confirmed no differences in mutant protein expression compared to wild-type protein expression in heterozygous MEFs. The R117C mutation has been identified in Li-Fraumeni-Like patients with increased cancer susceptibility. (J:325969)