Targeted nucleotide exchange in the alkaline phosphatase gene of HuH‐7 cells mediated by a chimeric RNA/DNA oligonucleotide

Although a variety of methods has been devised for modification of hepatic genes, none has been effective for long‐term correction of genetic disorders. In this study, we employed a recently described novel experimental strategy for site‐directed nucleotide exchange in genomic DNA of HuH‐7 human hepatoma cells. A chimeric 2′‐O‐methylated‐RNA/DNA oligonucleotide containing sequences complementary to 25 bases of the alkaline phosphatase gene was constructed as a duplex containing a G to A substitution at nucleotide 935. Cells were transfected with oligonucleotides for 48 hours, then harvested for DNA isolation and polymerase chain reaction (PCR) amplification of exon 6 of the alkaline phosphatase gene. Colony lifts were hybridized to 17 mer 32 P‐labeled oligonucleotide probes specific to the 935‐G and 935‐A sequences. Hybridizing colonies were grown, plasmid DNA isolated, and sequenced. Transfection efficiency was determined at 24 hours by nuclear uptake of fluorescein‐12‐dUTP‐labeled chimeric oligonucleotides. Colonies hybridizing with the 935‐A probe were identified only from cells transfected with the specific chimeric oligonucleotide; and there was no evidence of cross‐hybridization. Conversion of G to A at nucleotide 935 occurred at an overall frequency of up to 11.9% and when corrected for transfection efficiency approached 43%. No other alterations were detected in the sequence of exon 6 with the targeted nucleotide exchange. These results show that a single base pair alteration in the alkaline phosphatase gene of HuH‐7 cells can be introduced at a relatively high frequency following transfection with chimeric RNA/DNA oligonucleotides. This technique offers a novel and potentially powerful strategy for site‐directed hepatic gene alteration without the use of viral‐based vectors.