Efficient introduction of specific TP53 mutations into mouse embryonic fibroblasts and embryonic stem cells

Developers

Quan-Xiang Wei, Franciscus van der Hoeven, Monica Hollstein, Adam F Odell, etc.

Description of the technology

Clinical studies to assess the phenotypic impact of different classes of Trp53 tumor mutations (Trp53 codes p53 protein − tumor suppressor) are difficult because of the heterogeneity of tumors and the genetic diversity of human populations. In order to reduce genetic noise most relevant approach is in vitro studies with isogenic cell lines or p53-null cell lines transfected with plasmids expressing different mutants.

This technology provides a rapid, precise method for generating sets of embryonic stem cells or mouse embryonic fibroblasts harboring point mutations in Trp53 gene. The strategy uses cells from novel mouse strain − 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 mouse embryonic fibroblast 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 mouse embryonic fibroblasts. A library of cell lines expressing various p53 mutants derived from the same population of primary fibroblasts or embryonic stem cells can be acquired and screened in less than 1 month.

Practical application

The technology allows to study mutant phenotypes in vitro using the mouse embryonic fibroblast and embryonic stem cell systems. Additional applications could include the following.

Mating platform mice with other transgenics can be used as an efficient way to investigate how specific genetic defects in other cancer genes/pathways affect mutant p53 behavior.

These mutant ES cell lines may be used to generate new p53 mutant strains in a more cost-effective manner.

It is possible that other primary cells from platform mice have potential as starting material for TOP integration and generation of clones.

Any sequence of interest (e.g., a transgene, with promoter and full coding sequence), if flanked by attP sites, can be integrated into the platform site.

Conditional p53 alleles can be generated (modifications of the TOP plasmid: loxP, fluorescent tag, inducible on-off systems). This would be useful in the generation of mouse embryonic fibroblast lines retaining wild-type p53 function.

Laboratories

• Genetic Alterations in Carcinogenesis (C016), German Cancer Research Center (Deutsches Krebsforschungszentrum), Heidelberg (Germany)
• Transgenic Service (W450), German Cancer Research Center (Deutsches Krebsforschungszentrum), Heidelberg (Germany)
• Faculty of Medicine and Health, Leeds Institute of Genetics, Health and Therapeutics (LIGHT) Laboratories, University of Leeds, Leeds (UK)

Links

Publications

• Wei, Q.X. et al. «Efficient introduction of specific TP53 mutations into mouse embryonic fibroblasts and embryonic stem cells." 7 Nature Protocols (2012): 1145–1160.
• Wei, Q.X. et al. «Rapid derivation of genetically related mutants from embryonic cells harboring a recombinase-specific Trp53 platform." 10 Cell Cycle (2011): 1261–1270.
• Odell, A., Askham, J., Whibley, C. & Hollstein, M. «How to become immortal: let MEFs count the ways." 2 Aging (Albany NY) (2010): 160–165.