利用结构优化的TALE-TF高效诱导MEF细胞内源基因Oct4的表达（英文）

发布时间：2018-06-21 10:37

本文选题：TALE-N端 + 转录激活子样效应因子核酸酶(TALENs)　；参考：《中国生物化学与分子生物学报》2017年05期

【摘要】：转录激活子样效应因子核酸酶(TALENs)已用于基因组编辑,而TALE转录激活因子(TALE-TFs)可用于内源基因的调控。通过修饰的TALE蛋白与内源基因的启动子特异性结合来激活或抑制基因的表达。但是,对于截短了N端的TALE是否会影响TALENs的效率还不确定。本文设计了不同长度N端的TALENs哺乳动物及酵母表达载体,对应哺乳动物绿色荧光报告载体及酵母报告载体。分别在哺乳动物细胞和酵母细胞上对不同长度N端TALENs的活性进行了检测。检测结果发现,含120个氨基酸N端的TALEN在酵母AH109细胞中的活性最高,N端长度为153个氨基酸的TALENs在293T细胞中表现最高的活性,但N端长度为153、140和160个氨基酸的TALENs的活性差异不显著(P0.05)。随后,将结构优化了的TALE-TF用于小鼠胎儿成纤维细胞中激活Oct4基因。相对阴性对照组,TALE-TF将Oct4基因表达量上调了418倍。本研究结果表明,在哺乳动物细胞和酵母细胞中,具有最高活性TALENs的N端长度是不同的。本研究用结构优化的TALE-TF诱导内源基因高水平的表达,为从体细胞中获得iP S细胞提供了研究策略。
[Abstract]:Transcription-activator like effector nuclease (TALENs) has been used in genome editing, while the transcription-activator TALE-TFscan be used for the regulation of endogenous genes. Gene expression was activated or inhibited by specific binding of modified tar protein to the promoter of endogenous gene. However, it is not certain whether truncated N-terminal ale will affect the efficiency of TALENs. In this paper, we designed the mammalian and yeast expression vectors with different N-terminal length, corresponding to the mammalian green fluorescent report vector and yeast report vector. The activity of N-terminal TALENs with different lengths was detected in mammalian cells and yeast cells. The results showed that TALEN containing 120 amino acids showed the highest activity in yeast AH109 cells, and TALENs with 153 amino acids in N-terminal length showed the highest activity in 293T cells. However, the activity of TALENs with N-terminal length of 153140 and 160 amino acids was not significantly different from that of P0.05. Subsequently, TALE-TF with optimized structure was used to activate Oct4 gene in mouse fetal fibroblasts. TALE-TF increased Oct4 gene expression by 418 times. The results showed that the N-terminal length of TALENs with the highest activity was different in mammalian cells and yeast cells. In this study, the high level expression of endogenous gene was induced by TALE-TF, which provided a research strategy for obtaining IP S cells from somatic cells.
【作者单位】：西北农林科技大学动物科技学院;遵义师范学院生物与农业科技学院;
【基金】：Supported by Scientific and Technological Project of Shaanxi Province,China(No.2014K02-07-01) China Postdoctoral Science Foundation(No.2015M580887)~~
【分类号】：Q78

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