ausblenden:
Schlagwörter:
Amino Acid Sequence; Base Sequence; Binding Sites; Cell Line, Tumor; Chromosomes, Human/genetics; *DNA Repair; Deoxyribonucleases/*chemistry/*metabolism; Exons/genetics; Genes, p53/*genetics; Genetic Loci/genetics; HEK293 Cells; Humans; Introns/genetics; Models, Molecular; Molecular Sequence Data; Mutation; Plasmids/genetics; Reproducibility of Results; *Sequence Analysis; *Two-Hybrid System Techniques; *Zinc Fingers
Zusammenfassung:
The tumor suppressor gene p53 is mutated or deleted in over 50% of human tumors. As functional p53 plays a pivotal role in protecting against cancer development, several strategies for restoring wild-type (wt) p53 function have been investigated. In this study, we applied an approach using gene repair with zinc finger nucleases (ZFNs). We adapted a commercially-available yeast one-hybrid (Y1H) selection kit to allow rapid building and optimization of 4-finger constructs from randomized PCR libraries. We thus generated novel functional zinc finger nucleases against two DNA sites in the human p53 gene, near cancer mutation 'hotspots'. The ZFNs were first validated using in vitro cleavage assays and in vivo episomal gene repair assays in HEK293T cells. Subsequently, the ZFNs were used to restore wt-p53 status in the SF268 human cancer cell line, via ZFN-induced homologous recombination. The frequency of gene repair and mutation by non-homologous end-joining was then ascertained in several cancer cell lines, using a deep sequencing strategy. Our Y1H system facilitates the generation and optimisation of novel, sequence-specific four- to six-finger peptides, and the p53-specific ZFN described here can be used to mutate or repair p53 in genomic loci.
