title: Targeted nucleotide exchange in the alkaline phosphatase gene of HuH-7 cells mediated by a chimeric RNA/DNA oligonucleotide.
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.
authors.affiliation: Department of Medicine, University of Minnesota Medical School, Minneapolis 55455, USA.
authors.forename: B T
authors.initials: BT
authors.lastname: Kren
authors.forename: A
authors.initials: A
authors.lastname: Cole-Strauss
authors.forename: E B
authors.initials: EB
authors.lastname: Kmiec
authors.forename: C J
authors.initials: CJ
authors.lastname: Steer
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics*; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology; D002467:Cell Nucleus / Q000378:metabolism; D002678:Chimera; D004247:DNA / Q000235:genetics; D019793:Fluorescein; D005452:Fluoresceins; D018390:Gene Targeting; D005914:Globins / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000235:genetics*; D009841:Oligonucleotides / Q000235:genetics*; D011110:Polymorphism, Genetic; D012313:RNA / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D005452:Fluoresceins; D009711:Nucleotides; D009841:Oligonucleotides; D012313:RNA; D005914:Globins; D004247:DNA; D000469:Alkaline Phosphatase; D019793:Fluorescein