Engineered zinc finger nuclease-mediated homologous recombination of the human rhodopsin gene.
Engineered zinc finger nuclease-mediated homologous recombination of the human rhodopsin gene.
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工程锌指核酸酶介导的人视紫红质基因同源重组。
DOI：
10.1167/iovs.10-5781
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发表时间：
2010
影响因子：
4.4
通讯作者：
Kumar-Singh,Rajendra
中科院分区：
文献类型：
--
作者：
Greenwald,DavidL;Cashman,SiobhanM;Kumar-Singh,Rajendra
目的。目的：设计一种新的锌指核酸酶（ZFNs），以人类视紫红质基因为靶点，诱导供体DNA片段的同源重组。方法：根据已有的指南设计三指锌指核酸酶。为了检测ZFN的特异性，作者产生了稳定表达Pro23His视紫红质的人胚胎视网膜母细胞系，Pro23His视紫红质是北美常染色体显性视网膜色素变性最常见的突变。他们报道了这些视紫红质特异性zfn在人类基因组中诱导靶向双链断裂的定量，证明了它们诱导供体DNA片段同源重组的能力，并报道了zfn介导的同源重组频率的定量。与内源性同源重组相比，在人视紫红质基因中，zfn定向同源重组的绝对频率高达17%。结论：zfn是一种嵌合蛋白，在遗传性疾病的治疗中具有重要的潜力。在这项研究中，作者报道了针对人类视紫红质基因的新型ZFNs的设计。这些zfn可能有助于治疗视网膜疾病，如视网膜色素变性，这是发达国家遗传性失明的最常见原因之一。在此，他们还报道了供体片段设计和促进zfn介导的同源重组的体外条件的几个方面。
Purpose.: Novel zinc finger nucleases (ZFNs) were designed to target the human rhodopsin gene and induce homologous recombination of a donor DNA fragment.Methods.: Three-finger zinc finger nucleases were designed based on previously published guidelines. To assay for ZFN specificity, the authors generated human embryonic retinoblast cell lines stably expressing a Pro23His rhodopsin, the most common mutation associated with autosomal dominant retinitis pigmentosa in North America. They report quantification of these rhodopsin-specific ZFNs to induce a targeted double-strand break in the human genome, demonstrate their ability to induce homologous recombination of a donor DNA fragment, and report the quantification of the frequency of ZFN-mediated homologous recombination.Results.: Compared with endogenous homologous recombination, the authors observed a 12-fold increase in homologous recombination and an absolute frequency of ZFN-directed homologous recombination as high as 17% in the human rhodopsin gene.Conclusions.: ZFNs are chimeric proteins with significant potential for the treatment of inherited diseases. In this study, the authors report the design of novel ZFNs targeting the human rhodopsin gene. These ZFNs may be useful for the treatment of retinal diseases such as retinitis pigmentosa, one of the most common causes of inherited blindness in the developed world. Herein, they also report on several aspects of donor fragment design and in vitro conditions that facilitate ZFN-mediated homologous recombination.