Simultaneous targeted alteration of the tyrosinase and c-kit genes by single-stranded oligonucleotides

被引:40
作者
Alexeev, V
Igoucheva, O
Yoon, K
机构
[1] Thomas Jefferson Univ, Jefferson Med Coll, Dept Dermatol & Cutaneous Biol, Jefferson Inst Mol Med, Philadelphia, PA 19107 USA
[2] Thomas Jefferson Univ, Jefferson Med Coll, Dept Mol Pharmacol & Biochem, Philadelphia, PA 19107 USA
关键词
c-kit; tyrosinase; gene alteration; single-stranded oligonucleotides; albino metanocytes;
D O I
10.1038/sj.gt.3301862
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We have shown that various forms of oligonucleotides, chimeric RNA-DNA oligonucleotide (RDO) and single-stranded oligodeoxynucleotide (ODN), are capable of chromosomal gene alterations in mammalian cells. Using two ODNs we corrected an inactivating mutation in the tyrosinase gene and introduced an activating mutation into the c-kit gene in a single albino mouse melanocyte. Relying on a pigmentation change caused by tyrosinase gene correction, we determined the frequency of gene targeting events ranging from 2 x 10(-4) to 1 X 10(-3), which is comparable to our previously published data using RDO. However, ODN showed more reproducible gene correction than RDO and produced pigmented cells among 60% of experiments, in comparison with 10% by RDO. DNA sequence analysis of the converted cells revealed that two out of eight individual pigmented clones harbored the mutated c-kit gene. Targeted modification of both genes resulted in the ability of the tyrosinase to convert tyrosine to melanin, and in the constitutive activation of the Kit receptor kinase. Thus, for the first time, we demonstrate the feasibility of simultaneous targeting of two genes in a single cell and show that a selection strategy to identify cells that have undergone a gene modification can enrich the targeted cells with the desired gene alteration.
引用
收藏
页码:1667 / 1675
页数:9
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