Stable and inheritable changes in genotype and phenotype of albino melanocytes induced by an RNA-DNA oligonucleotide

• Published in: Nature biotechnology 1998-12, Vol.16 (13), p.1343-1346
• Main Authors: Yoon, Kyonggeun, Alexeev, Vitali
• Format: Article
• Language: English
• Subjects: Agriculture; Amino Acid Sequence; Animals; Base Sequence; Bioinformatics; Biological and medical sciences; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; Cell Line; Diverse techniques; DNA - chemistry; DNA - metabolism; Fundamental and applied biological sciences. Psychology; Genotype; Life Sciences; Melanocytes - metabolism; Mice; Molecular and cellular biology; Molecular Sequence Data; Monophenol Monooxygenase - genetics; Nucleic Acid Hybridization; Phenotype; Pigmentation - genetics; Point Mutation; research-article; RNA - chemistry; RNA - metabolism
• ISSN: 1087-0156; 1546-1696
• DOI: 10.1038/4322

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 (T G T→T C T) 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.