Alexeev V, Yoon K
Department of Dermatology and Cutaneous Biology, Jefferson Molecular Institute of Medicine, Thomas Jefferson University, and Jefferson Medical College, Philadelphia, PA 19107, USA.
Nat Biotechnol. 1998 Dec;16(13):1343-6. 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 (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.
已开发出实验策略，利用由RNA和DNA组成的嵌合寡核苷酸来校正点突变。我们使用这些RNA-DNA寡核苷酸来校正小鼠酪氨酸酶中的一个点突变，酪氨酸酶是黑色素合成和色素沉着的关键酶。源自白化病小鼠的黑素细胞在酪氨酸酶基因中含有纯合点突变（TGT→TCT），导致氨基酸从半胱氨酸变为丝氨酸。校正这个点突变会导致酪氨酸酶活性和黑色素合成的恢复，从而改变细胞的色素沉着。将RNA-DNA寡核苷酸转染到白化病黑素细胞后，我们检测到了黑色色素沉着的细胞并分离出多个单克隆。所有黑色色素沉着的克隆在酪氨酸酶基因的单个等位基因中都表现出点突变的校正。通过抗酪氨酸酶抗体检测到全长酪氨酸酶，并且在所有转化的黑色色素沉着克隆中酶活性都得到了恢复。由于突变型酪氨酸酶的蛋白水解切割，在白化病细胞中仅检测到降解片段。转化的黑色色素沉着克隆的表型和基因型是稳定的。这些结果通过克隆分析证明了RNA-DNA寡核苷酸在基因组序列、蛋白质和表型变化水平上的永久和稳定的基因校正。
