FIG. 7.

Repair of a point mutation in an endogenous locus (DA5 cell line). (A) Scheme of HpE6rep. One repair-PPRH (HpE6rep) was designed against the exon 6 of the dhfr gene. HpE6rep contained a single extended sequence of DNA bearing the deleted nucleotide, and three pyrymidine interruptions in the hairpin core complementary to the target sequence. (B) DHFR mRNA levels in repaired DA5 cells. RNA was extracted from surviving DA5 cell colonies upon transfection with HpE6rep. DHFR mRNA levels were determined using qRT-PCR and were normalized to APRT. DHFR mRNA levels of the repaired colonies were referred to the positive control UA21. (C) DHFR protein levels in repaired DA5 cells. Total protein extracts were obtained from surviving DA5 cell colonies upon transfection with HpE6rep. Western blots were performed with 100 μg of total extracts and were normalized to tubulin levels. Protein levels of the repaired colonies were referred to the protein levels of the positive control UA21. A representative image of the Western analyses is shown. Color images available online at www.liebertpub.com/hgtb