Trp53^tm1Glo

A two-step procedure was employed to introduce an arginine to histidine substitute at residue 172 (R172H), the equivalent of R175H in humans. First, a neo-derived cassette replaced exons 2-6 by homologous recombination. Next, a vector containing exons 2-6 and carrying a CGC to CAC missense mutation at codon 172 was used to replace the neo cassette and reconstitute the gene. SSCP confirmed successful integration of the mutant allele and restriction digest analysis confirmed reconstitution of the gene. Immunoprecipitation experiments with mouse embryonic fibroblasts derived from homozygous mutant embryos showed that mutant protein is expressed at a level similar to wild-type. RT-PCR analysis detected full-length transcript and 2 mutant transcripts involving exon 3 deletions due to a mutation at the splice acceptor site of intron 2. (J:61683)