FIG. 1.
Targeted disruption of the mTOR gene by the cre-loxP strategy. (A) A neomycin resistance cassette (neo) flanked by loxP sequences and a thymidine kinase expression cassette (tk) were introduced in the genomic mTOR clone for selection of ES cells. A loxP site was inserted upstream of the presumptive promoter sequence. (B) Southern blot analysis of WT and mTORneo ES cell DNAs digested with NheI and hybridized with the 5′ probe indicated in the mTOR gene map. (C) Diagram of cre-mediated full excision of the mTORneo allele. The mTOR coding sequence deleted by recombination is indicated by the first and last corresponding amino acids (Met1 and Arg260). AC16, AC14, and AC11 correspond to the oligonucleotide primers used for genotyping the WT and mTOR deletion alleles. (D) Genotyping of mouse DNA was performed by PCR with a mixture of three primers (see Materials and Methods). PCR amplification with primers AC14 and AC11 produced a 273-bp DNA fragment (lower band) for the WT mTOR allele, while PCR amplification with primers AC16 and AC11 produced a 522-bp fragment (upper band) for the excised allele.