Alexeev V, Igoucheva O, Yoon K
Department of Dermatology and Cutaneous Biology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Jefferson Medical College, Philadelphia, PA, USA.
Gene Ther. 2002 Dec;9(24):1667-75. doi: 10.1038/sj.gt.3301862.
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.
我们已经表明，各种形式的寡核苷酸，即嵌合RNA-DNA寡核苷酸（RDO）和单链寡脱氧核苷酸（ODN），能够在哺乳动物细胞中引起染色体基因改变。我们使用两种ODN纠正了酪氨酸酶基因中的失活突变，并在一只白化病小鼠黑素细胞中向c-kit基因引入了激活突变。依靠酪氨酸酶基因纠正引起的色素沉着变化，我们确定了基因靶向事件的频率范围为2×10⁻⁴至1×10⁻³，这与我们之前使用RDO发表的数据相当。然而，与RDO相比，ODN显示出更可重复的基因纠正，并且在60%的实验中产生了色素沉着细胞，而RDO为10%。对转化细胞的DNA序列分析表明，八个单独的色素沉着克隆中有两个含有突变的c-kit基因。对这两个基因的靶向修饰导致酪氨酸酶将酪氨酸转化为黑色素的能力，并导致Kit受体激酶的组成型激活。因此，我们首次证明了在单个细胞中同时靶向两个基因的可行性，并表明一种识别经历了基因修饰的细胞的选择策略可以富集具有所需基因改变的靶向细胞。
