本文選題：TALE-N端 + 轉(zhuǎn)錄激活子樣效應(yīng)因子核酸酶(TALENs)　； 參考：《中國(guó)生物化學(xué)與分子生物學(xué)報(bào)》2017年05期

【摘要】：轉(zhuǎn)錄激活子樣效應(yīng)因子核酸酶(TALENs)已用于基因組編輯,而TALE轉(zhuǎn)錄激活因子(TALE-TFs)可用于內(nèi)源基因的調(diào)控。通過(guò)修飾的TALE蛋白與內(nèi)源基因的啟動(dòng)子特異性結(jié)合來(lái)激活或抑制基因的表達(dá)。但是,對(duì)于截短了N端的TALE是否會(huì)影響TALENs的效率還不確定。本文設(shè)計(jì)了不同長(zhǎng)度N端的TALENs哺乳動(dòng)物及酵母表達(dá)載體,對(duì)應(yīng)哺乳動(dòng)物綠色熒光報(bào)告載體及酵母報(bào)告載體。分別在哺乳動(dòng)物細(xì)胞和酵母細(xì)胞上對(duì)不同長(zhǎng)度N端TALENs的活性進(jìn)行了檢測(cè)。檢測(cè)結(jié)果發(fā)現(xiàn),含120個(gè)氨基酸N端的TALEN在酵母AH109細(xì)胞中的活性最高,N端長(zhǎng)度為153個(gè)氨基酸的TALENs在293T細(xì)胞中表現(xiàn)最高的活性,但N端長(zhǎng)度為153、140和160個(gè)氨基酸的TALENs的活性差異不顯著(P0.05)。隨后,將結(jié)構(gòu)優(yōu)化了的TALE-TF用于小鼠胎兒成纖維細(xì)胞中激活Oct4基因。相對(duì)陰性對(duì)照組,TALE-TF將Oct4基因表達(dá)量上調(diào)了418倍。本研究結(jié)果表明,在哺乳動(dòng)物細(xì)胞和酵母細(xì)胞中,具有最高活性TALENs的N端長(zhǎng)度是不同的。本研究用結(jié)構(gòu)優(yōu)化的TALE-TF誘導(dǎo)內(nèi)源基因高水平的表達(dá),為從體細(xì)胞中獲得iP S細(xì)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.
【作者單位】： 西北農(nóng)林科技大學(xué)動(dòng)物科技學(xué)院;遵義師范學(xué)院生物與農(nóng)業(yè)科技學(xué)院;
【基金】：Supported by Scientific and Technological Project of Shaanxi Province,China(No.2014K02-07-01) China Postdoctoral Science Foundation(No.2015M580887)~~
【分類號(hào)】：Q78

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