1] Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA [2] Department of Pediatrics, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA.
Mol Ther Nucleic Acids. 2013 Apr 23;2(4):e87. doi: 10.1038/mtna.2013.12.
The ability to direct human telomerase reverse transcriptase (hTERT) expression through either genetic control or tunable regulatory factors would advance not only our understanding of the transcriptional regulation of this gene, but also potentially produce new strategies for addressing telomerase-associated disease. In this work, we describe the engineering of artificial zinc finger transcription factors (ZFTFs) and ZF nucleases (ZFNs) to target sequences within the hTERT promoter and exon-1. We were able to identify several active ZFTFs that demonstrate a broadly tunable response when screened by a cell-based transcriptional reporter assay. Using the same DNA-binding domains, we generated ZFNs that were screened in combinatorial pairs in cell-based extrachromosomal single-strand annealing (SSA) assays and in gene-targeting assays using stably integrated constructs. Selected ZFN pairs were tested for the ability to induce sequence changes in a Cel1 assay and we observed frequencies of genomic modification up to 18.7% at the endogenous hTERT locus. These screening strategies have pinpointed several ZFN pairs that may be useful in gene editing of the hTERT locus. Our work provides a foundation for using engineered ZF proteins (ZFPs) for modulation of the hTERT locus.Molecular Therapy - Nucleic Acids (2013) 2, e87; doi:10.1038/mtna.2013.12; published online 23 April 2013.
通过遗传控制或可调调控因子来指导人端粒酶逆转录酶（hTERT）的表达能力不仅将推进我们对该基因转录调控的理解，而且还可能为解决端粒酶相关疾病产生新的策略。在这项工作中，我们描述了人工锌指转录因子（ZFTF）和锌指核酸酶（ZFN）的工程设计，以针对 hTERT 启动子和外显子 1 内的序列。我们能够鉴定出几种活性 ZFTF，当通过基于细胞的转录报告基因测定筛选时，它们表现出广泛可调的反应。使用相同的 DNA 结合结构域，我们生成了 ZFN，它们在基于细胞的染色体外单链退火（SSA）测定和使用稳定整合构建体的基因靶向测定中以组合对进行筛选。选择的 ZFN 对用于在 Cel1 测定中诱导序列变化的能力进行了测试，我们观察到在天然 hTERT 基因座中基因组修饰的频率高达 18.7％。这些筛选策略已经确定了几种 ZFN 对，它们可能在 hTERT 基因座的基因编辑中有用。我们的工作为使用工程化 ZF 蛋白（ZFPs）来调节 hTERT 基因座提供了基础。分子治疗 - 核酸（2013）2，e87; doi:10.1038/mtna.2013.12; 在线发表 2013 年 4 月 23 日。
Zotero 插件
