title: Rapid derivation of genetically related mutants from embryonic cells harboring a recombinase-specific Trp53 platform.
abstract: Recombinase-mediated cassette exchange (RMCE) is a powerful method for achieving gene targeting repeatedly at a single mammalian locus. This approach could be applied to the efficient establishment of genetically related cell lines harboring different p53 mutations found in human tumors. To this end we generated a mouse strain called p53 Platform mice (PLF mice), containing PhiC31 integrase-specific attP sequences at the Trp53 locus. The attP sites flank a PGK-neo cassette that has replaced exons 2 to 9 of the endogenous murine p53 gene, generating a null allele. Electroporation of a fluorescence indicator plasmid into embryonic stem (ES) cell lines from PLF mice demonstrated that PhiC31 integrase-mediated cassette exchange (IMCE) can be achieved at > 60% efficiency without selecting against random insertion. To produce somatic cell lines with endogenously controlled expression of mutant p53, we performed IMCE in PLF murine embryonic fibroblasts (MEFs) with plasmid constructs containing human p53 gene sequences carrying specific tumor-associated missense mutations (A138V; G245S). The MEF cell lines produce the expected mutated mRNA transcripts and express p53 protein that is phosphorylated at serine 15 following DNA damage. Within a few weeks one can thus acquire a family of p53 mutant cell lines from the same population of primary cells, but each harboring a different mutation.
authors: Genetic Alterations in Carcinogenesis (C016), German Cancer Research Center, Heidelberg, Germany.
authors: Quan-Xiang
authors: QX
authors: Wei
authors: Adam F
authors: AF
authors: Odell
authors: Franciscus
authors: F
authors: van der Hoeven
authors: Monica
authors: M
authors: Hollstein
mesh_terms: D000483:Alleles; D000818:Animals; D001483:Base Sequence; D002461:Cell Line, Transformed; D004249:DNA Damage; D018274:Electroporation; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005786:Gene Expression Regulation; D018390:Gene Targeting / Q000379:methods*; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D020033:Protein Isoforms / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D045522:Recombinases / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D020033:Protein Isoforms; D012333:RNA, Messenger; D045522:Recombinases; D016159:Tumor Suppressor Protein p53; D019426:Integrases