Low-dose bleomycin induces targeted gene repair frequency in cultured melan-c cells using chimeric RNA/DNA oligonucleotide transfection

Abstract

We attempted to raise the frequency of targeted gene repair attained by RNA/DNA chimeraplasty through activation of endogenous DNA recombination/repair system in cultured melan-c cells. For elevating the repair activity, either gamma-ray irradiation at 2 Gy or bleomycin addition at 5 nM to the culture medium was done. Subsequently, the tyrosinase-targeted chimeric RNA/DNA oligonucleotides were transfected. The frequency of targeted gene repair was assessed by three means, i.e., changes in cell color from transparent to dark black, restriction fragment band patterns, and DNA sequencing analysis. The results indicated that the gamma-ray irradiation did not improve the efficiency at all. In contrast, the low-dose bleomycin addition induced the frequency of targeted base conversion, compared with transfection with chimeric RDOs alone, although the frequency was still insufficient and varied from 0.1 to 10% of the total cultured cells.