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. 2011 Jun 9;6(6):e20913. doi: 10.1371/journal.pone.0020913

Figure 5. Targeted genomic recombination with p53 donor plasmid.

Figure 5

(A) Schematic of the genomic p53 locus and the donor plasmid, with modified DNA sequences at z771 and z1166 target sites (“barcodes”; highlighted in red). Barcodes allow selective PCR but do not change the p53 protein sequence. Black arrows indicate primers for genomic PCR that hybridize to the chromosomal p53 locus outside of the region corresponding to donor sequence. (B) Targeted donor plasmid recombination into the p53 z771 and z1166 loci in HEK293T cells. ZFN-induced recombination is shown by semi-nested PCR, on purified external genomic PCR template, with forward primers that discriminate between wild-type and barcode sequence. (C) Targeted donor recombination at the p53 z1166 locus in human SF268 glioblastoma cells. (D) Restoration of p53 wild-type sequence at codon 273 in ZFP-treated SF268 glioblastoma cells. The relative positions of the z1166 cutting site and codon 273 are indicated in Fig. 1B.