[Construction of a SV40 promoter specific artificial transcription factor].
[Construction of a SV40 promoter specific artificial transcription factor].
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SV40启动子特异性人工转录因子的构建
DOI：
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发表时间：
2003
期刊：
影响因子：
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通讯作者：
Pei
中科院分区：
文献类型：
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作者：
Xinghui Zhao;Xudong Zhu;Juan Liu;Xiang;Pei
转录受转录因子调节。天然转录因子通常由至少两个功能结构域组成：DNA结合结构域和效应子结构域。据此，新的人工转录因子被设计为上调或下调靶基因的转录和表达。Cys 2-His 2锌指结构域是一种DNA结合模块，已被广泛用作人工转录因子中的DNA结合结构域。每个锌指结构域包含约30个通过螯合锌离子而采用紧凑结构的氨基酸，通常通过结合DNA序列的3个碱基对来发挥功能。连接在一起的几个锌指将按比例结合更长的DNA序列。根据“二分互补”文库策略，构建了一对锌指噬菌体展示文库。构建文库后，选择SV 40启动子上的9 bp序列（5 '-GCAGAGGCC-3'）作为下一步的靶序列。在平行选择、PCR扩增、所需片段回收、再连接和另外几轮选择后，进行噬菌体酶联ELISA实验以鉴定显示具有预定序列特异性的锌指的特异性结合克隆。然后选择具有强ELISA信号的两个克隆以测试与其完整靶位点（5 '-GCAGAGGCC-3'）和含有单个转换突变的位点的结合。两个克隆之一（克隆3）的结合特异性显示相当好。将靶向SV 40启动子的三指DNA结合结构域（即克隆3上的锌指序列）与KOX 1抑制结构域KRAB融合并克隆入pcDNA3.1（+）（其表达产物为人工转录因子）中。锌指（其表达产物是人工转录因子的DNA结合结构域）和仅KRAB结构域（其表达产物是人工转录因子的效应子结构域）也分别克隆到同一表达载体中。所有构建体均含有N-末端核定位信号。用pGL 3-Control和pRL-TK共转染各载体（包括不含插入序列的pcDNA3.1（+）），以荧光素酶活性为指标检测转染产物的功能。我们的人工转录因子能有效抑制报告基因的表达，而只有DNA结合结构域或效应结构域的抑制作用不明显。人工转录因子通过添加不同的效应子结构域和改变DNA结合结构域，具有广泛的应用前景。
Transcriptions are regulated by transcription factors. Natural transcription factors usually consist of at least two functional domains: a DNA-binding domain and an effector domain. According to this, novel artificial transcription factors are designed to up or down regulate transcription and expression of a target gene. The Cys2-His2 zinc finger domain is a DNA-binding module that has been widely used as the DNA-binding domain in artificial transcription factors. Each zinc finger domain, which comprises about 30 amino acids that adopt a compact structure by chelating a zinc ion, typically functions by binding 3 base pairs of DNA sequence. Several zinc fingers linked together would bind proportionally longer DNA sequences. According to the "bipartite complementary" library strategy, a pair of zinc finger phage display libraries were constructed. After construction of the libraries, a 9bp sequence (5'-GCAGAGGCC-3') on the promoter of SV40 was chosen as a target for next step. After parallel selection, PCR amplification, desired fragments recovery, re-ligation, and additional rounds of selection, phage enzyme-linked ELISA experiments were performed to identify specific binding clones displaying the zinc fingers with predetermined sequence-specificity to our target sequence. Then two clones with strong ELISA signals were chosen to be tested for binding both to its full target site (5'-GCAGAGGCC-3') and to sites containing single transition mutations. The binding specificity of one of the two clones (clone 3) was shown to be fairly good. The three-finger DNA-binding domain targeted to SV40 promoter, that is, zinc finger sequences on clone 3, was fused to KOX1 suppression domain KRAB and cloned into pcDNA3.1 (+) (which expression product was artificial transcription factor). The zinc fingers (which expression product was the DNA-binding domain of artificial transcription factor) and KRAB domain only (which expression product was effector domain of artificial transcription factor) were also cloned separately into the same expression vector. All constructs contained an N-terminal nuclear localization signal. Every of the vectors (including pcDNA3.1 (+) without inserting sequences) were cotransfected with pGL3-Control and pRL-TK and the activity of luciferase was used to indicate the function of product from transfected expression vectors. Our artificial transcription factor was proved to repress the expression of reporter gene efficiently,while with only DNA-binding domain or effector domain the repression was not remarkable. By adding different effector domains and changing the DNA-binding domain, artificial transcription factor would have a wide range of potential applications.