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Author:
Date:
2015-07-20
[Abstract] Transcription activator-like effectors (TALEs) are naturally occurring proteins secreted by the plant pathogen, Xanthomonas, and fused to the Fok1 endonuclease to generate TALE nucleases (TALENs). TALEN pairs bind to specific DNA sequences initiating Fok1 dimerization and double-stand cleavage of DNA within the TALEN target site. This cleavage event triggers cellular repair mechanisms that result in insertions and/or deletions (indels), which enable gene knockout. The high specificity and efficiency of TALENs makes them important tools for genome editing. Here, we describe a method for the generation of single-cell clones with targeted gene knockout by TALEN using co-transfection and FACS with a fluorescent reporter. This protocol was designed to knockout cell death-inducing ...
[摘要] 转录激活子样效应器（TALE）是由植物病原体黄单胞菌分泌的天然存在的蛋白质，并且与Fok1内切核酸酶融合以产生TALE核酸酶（TALEN）。 TALEN对与特异性DNA序列结合，启动FAL1二聚化和在TALEN靶位点内双链切割DNA。 该切割事件触发导致插入和/或缺失（插入缺失）的细胞修复机制，其使得能够进行基因敲除。 TALEN的高特异性和高效性使其成为基因组编辑的重要工具。 在这里，我们描述了使用共转染和FACS与荧光报告基因通过TALEN产生具有靶向基因敲除的单细胞克隆的方法。 该方案设计为在Huh7.5细胞中敲除诱导细胞死亡的DFFA样效应子b，CIDEB; 然而，该协议可以应用于广泛的细胞类型和感兴趣的基因。
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