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

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Abstract
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 32P-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.

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