Different frequency of gene targeting events by the RNA-DNA oligonucleotide among epithelial cells.
Different frequency of gene targeting events by the RNA-DNA oligonucleotide among epithelial cells.
复制标题
上皮细胞中 RNA-DNA 寡核苷酸的基因靶向事件的不同频率。
DOI：
10.1046/j.1523-1747.1998.00403.x
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Yoon,K
中科院分区：
文献类型：
--
作者：
Santana,E;Peritz,AE;Iyer,S;Uitto,J;Yoon,K
一种由RNA和DNA组成的独特的杂交寡核苷酸已被证明可以在染色体外和染色体靶标中以定点特异和可遗传的方式纠正点突变。为了开发新的皮肤基因疗法，我们测试了两种寡核苷酸，它们被证明在几种上皮细胞类型中产生了碱性磷酸酶和β-珠蛋白基因的点突变。两种RNA-DNA寡核苷酸对高度转化的上皮细胞(HeLa)的转化率均为5%。相比之下，其他永生化上皮细胞(HaCaT)或人原代角质形成细胞在限制性片段长度多态性分析中没有表现出任何可检测到的基因转换水平，表明转换频率不到1%。用放射性标记的寡核苷酸或荧光素标记的寡核苷酸测量，HeLa细胞细胞核中的寡核苷酸浓度与HaCaT或人原代角质形成细胞的浓度相似。此外，RNA-DNA寡核苷酸在细胞核内表现出较长的稳定性。因此，在我们的实验条件下，寡核苷酸的摄取和核稳定似乎都不是基因靶向事件的限制因素。这些结果表明基因打靶的频率在不同的细胞中是不同的，这表明细胞重组和DNA修复活动可能是重要的。
A unique hybrid oligonucleotide composed of both RNA and DNA has been shown to correct a point mutation in a site-specific and inheritable manner in extrachromosomal and chromosomal targets. In order to develop new gene therapeutics for skin, we tested two oligonucleotides that were shown to create a point mutation in alkaline phosphatase and β-globin genes in several epithelial cell types. Highly transformed epithelial cells (HeLa) exhibited a conversion frequency of 5% by both RNA-DNA oligonucleotides. In comparison, other immortalized epithelial cells (HaCaT) or human primary keratinocytes did not show any detectable level of gene conversion by the restriction fragment length polymorphism analysis, indicating less than 1% conversion frequency. The concentration of the oligonucleotide in the nuclei of HeLa cells was similar to that of HaCaT or human primary keratinocytes measured by a radio-labeled or a fluorescein-conjugated oligonucleotide. Moreover, the RNA-DNA oligonucleotide exhibited a prolonged stability in the nucleus. Thus, neither uptake nor nuclear stability of the oligonucleotide appears to be a limiting factor in gene targeting events under our experimental conditions. These results indicate that the frequency of gene targeting varies among different cells, suggesting that cellular recombination and DNA repair activities may be important.