Stable and inheritable changes in genotype and phenotype of albino melanocytes induced by an RNA-DNA oligonucleotide.
Experimental strategies have been developed to correct point mutations using chimeric oligonucleotides composed of RNA and DNA. We used these RNA-DNA oligonucleotides to correct a point mutation in mouse tyrosinase, a key enzyme for melanin synthesis and pigmentation. Melanocytes derived from albino mice contain a homozygous point mutation (TGT-->TCT) in the tyrosinase gene, resulting in an amino acid change from Cys-->Ser. Correction of this point mutation results in the restoration of tyrosinase activity and melanin synthesis, thus changing the pigmentation of the cells. Upon transfection of the RNA-DNA oligonucleotide to albino melanocytes, we detected black-pigmented cells and isolated multiple single clones. All black-pigmented clones exhibited a correction of the point mutation in a single allele of the tyrosinase gene. A full-length tyrosinase was detected by an antityrosinase antibody, and the enzymatic activity was restored in all converted black-pigmented clones. Only degraded fragments were detected in albino cells due to proteolytic cleavage of mutant tyrosinase. The phenotype and genotype of converted black-pigmented clones was stable. These results demonstrate a permanent and stable gene correction by the RNA-DNA oligonucleotide at the level of genomic sequence, protein, and phenotypic change by clonal analysis.
Alexeev
V
V
Department of Dermatology and Cutaneous Biology, Jefferson Molecular Institute of Medicine, Thomas Jefferson University, and Jefferson Medical College, Philadelphia, PA 19107, USA.
Yoon
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D005838:Genotype; D008544:Melanocytes / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D010641:Phenotype; D010858:Pigmentation / Q000235:genetics; D017354:Point Mutation; D012313:RNA / Q000737:chemistry / Q000378:metabolism*
D012313:RNA; D004247:DNA; D014442:Monophenol Monooxygenase