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）  

An Efficient Method to Induce Endogenous Oct4 Expression in MEF Cells by Optimized Design of TALE-TF

作　　者：吴芸[1,2] 张志强[1] 李铎[1] 徐坤[1] 韩芙蓉[1] 任充华[1] 闫强[1] 王昕[1] 张智英[1] 

机构地区：[1]西北农林科技大学动物科技学院,陕西杨凌712100 [2]遵义师范学院生物与农业科技学院,贵州遵义563000

出　　处：《中国生物化学与分子生物学报》2017年第5期461-469,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：Supported by Scientific and Technological Project of Shaanxi Province,China(No.2014K02-07-01);China Postdoctoral Science Foundation(No.2015M580887)~~

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(P>0.05)。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。The TALE-based nucleases （TALENs） and transcript factors （TALE-TFs） have been used for genome editing and targeting endogenous gene regulation. Moreover, TALE proteins can be modified to activate gene expression by targeting its enhancer. However, the optimal length of TALE N-terminus that may affect the cleavage efficiency of TALENs is uncertain. In this study, we design N-terminal targeting strategy to achieve higher cleavage efficiency by optimizing the length of N-terminus using our reporter systems in both yeast and mammalian cell lines. For this purpose we first constructed a series of truncated mutants in the N-terminal sequences of TALENs expression plasmids, and yeast and mammalian reporter systems, respectively. Gene transfer and activation efficiency analyses showed that the truncated sequence with 120 N-terminal residues produced the highest efficiency in yeast AH109 cells, while that of 153 N-terminal residues gave the highest efficiency in 293T ceils, but no significant difference in the efficiency among the 153, 140 and 160 N-terminal residues （P 〉 0. 05）. Subsequently, we used the optimized N-terminal structure for TALE-TF to activate the stringently silenced pluripotent gene Oct4 in mouse embryonic fibroblasts （MEFs） , where the expression of the endogenous Oct4 gene that plays a crucial role in the generation of induced pluripotent stem cells （iPSCs） from somatic cells was enhanced up to 418-fold, compared with the negative control. Our results suggest that the length of the N-terminal sequence of TALENT required for its optimal function is different between yeast and mammalian cells. Our data also provide an optimization strategy by modifying the TALE-TF to induce endogenous gene expression at high levels, which is perhaps significant for generation of iPS cells from somatic cells.

关 键 词：TALE-N端 转录激活子样效应因子核酸酶(TALENs) Oct4基因 TALE转录激活因子(TALE-TF) 

分 类 号：Q786[生物学—分子生物学]