Abstract
This protocol describes a rapid, precise method for generating sets of embryonic stem (ES) cells or mouse embryonic fibroblasts (MEFs) harboring point mutations in the p53 tumor suppressor gene (officially known as Trp53). The strategy uses cells from the Trp53 (p53-null) 'platform' mouse, which allows site-specific integration of plasmid DNA into the Trp53 locus. Simple PCR protocols identify correctly targeted clones and immunoblots verify re-expression of the protein. We also present protocol modifications needed for efficient recovery of MEF clones expressing p53 constructs that retain wild-type function, including growth at low (3%) oxygen and transient downregulation of p53 regulators to forestall cell senescence of primary MEFs. A library of cell lines expressing various p53 mutants derived from the same population of primary fibroblasts or platform ES cells can be acquired and screened in less than 1 month.