Dissection of splicing regulation at an endogenous locus by zinc-finger nuclease-mediated gene editing.
Dissection of splicing regulation at an endogenous locus by zinc-finger nuclease-mediated gene editing.
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DOI：
10.1371/journal.pone.0016961
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发表时间：
2011-02-08
期刊：
影响因子：
3.7
通讯作者：
Cost GJ
中科院分区：
文献类型：
--
作者：
Cristea S;Gregory PD;Urnov FD;Cost GJ
由于传统的靶向突变的巨大困难，RNA剪接调控序列难以原位研究。锌指核酸酶（ZFN）技术允许将位点特异性突变快速有效地引入哺乳动物染色体中。使用ZFN对沿着供体质粒来操纵DNA修复的结果，我们在中国仓鼠卵巢细胞中的内源性BAX基因的第四内含子中引入了几个离散的靶向突变。靶向假定的lymphocyte分支点、多聚嘧啶段和剪接受体位点。我们回收了许多携带预期突变的其他同基因克隆，并分析了每个克隆对BAX前mRNA剪接的影响。突变的三个可能的分支点之一，多聚嘧啶道，和剪接受体位点都导致从BAX mRNA中排除外显子5。有趣的是，这些外显子跳跃突变允许使用内含子4内的隐蔽剪接受体位点。这些数据表明，ZFN介导的基因编辑是一种非常有效的工具，用于在其内源性背景下解剖前体mRNA剪接调控序列。
Sequences governing RNA splicing are difficult to study in situ due to the great difficulty of traditional targeted mutagenesis. Zinc-finger nuclease (ZFN) technology allows for the rapid and efficient introduction of site-specific mutations into mammalian chromosomes. Using a ZFN pair along with a donor plasmid to manipulate the outcomes of DNA repair, we introduced several discrete, targeted mutations into the fourth intron of the endogenous BAX gene in Chinese hamster ovary cells. Putative lariat branch points, the polypyrimidine tract, and the splice acceptor site were targeted. We recovered numerous otherwise isogenic clones carrying the intended mutations and analyzed the effect of each on BAX pre-mRNA splicing. Mutation of one of three possible branch points, the polypyrimidine tract, and the splice acceptor site all caused exclusion of exon five from BAX mRNA. Interestingly, these exon-skipping mutations allowed usage of cryptic splice acceptor sites within intron four. These data demonstrate that ZFN-mediated gene editing is a highly effective tool for dissection of pre-mRNA splicing regulatory sequences in their endogenous context.
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影响因子：
14.9
作者：
Orlando SJ;Santiago Y;DeKelver RC;Freyvert Y;Boydston EA;Moehle EA;Choi VM;Gopalan SM;Lou JF;Li J;Miller JC;Holmes MC;Gregory PD;Urnov FD;Cost GJ
通讯作者：
Cost GJ
影响因子：
64.8
作者：
HORNIG, H;AEBI, M;WEISSMANN, C
通讯作者：
WEISSMANN, C
影响因子：
46.9
作者：
Hockemeyer, Dirk;Soldner, Frank;Beard, Caroline;Gao, Qing;Mitalipova, Maisam;DeKelver, Russell C.;Katibah, George E.;Amora, Ranier;Boydston, Elizabeth A.;Zeitler, Bryan;Meng, Xiangdong;Miller, Jeffrey C.;Zhang, Lei;Rebar, Edward J.;Gregory, Philip D.;Urnov, Fyodor D.;Jaenisch, Rudolf
通讯作者：
Jaenisch, Rudolf
DOI：
10.1073/pnas.0800940105
发表时间：
2008-04-15
影响因子：
11.1
作者：
Santiago, Yolanda;Chan, Edmond;Collingwood, Trevor N.
通讯作者：
Collingwood, Trevor N.
影响因子：
14.9
作者：
Gao K;Masuda A;Matsuura T;Ohno K
通讯作者：
Ohno K