斑马鱼心脏发育候选基因Klhl31的TALEN打靶有效性分析

发布时间：2018-07-29 16:42

【摘要】：TALEN(transcription activator-like effector nuclease)打靶是一种定向敲除基因技术,利用该技术能解决目前在斑马鱼体内难以进行基因敲除的难题。Klhl31是本实验室鉴定的人类心脏发育候选基因,且目前国内外尚无斑马鱼Klhl31敲除品系。为了在斑马鱼的整体水平鉴定Klhl31的生物学功能,我们利用TALEN打靶技术建立斑马鱼Klhl31基因敲除品系。酶切验证的结果显示,注射了TALEN表达质粒的胚胎在酶切位点处发生了基因突变,该突变破坏了酶切位点导致酶切出现阳性结果,证明了TALEN打靶的有效性。对F0代突变体成鱼的筛选过程中,经酶切验证,同样出现了阳性结果。这些结果说明用TALEN这种技术成功敲除了斑马鱼Klhl31基因,获得了Klhl31基因敲除的嵌合体斑马鱼。
[Abstract]:TALEN (transcription activator-like effector nuclease) targeting is a directed knockout technique, which can be used to solve the difficult problem of gene knockout in zebrafish. Klhl31 is a candidate gene for human heart development identified by our laboratory. There are no zebrafish Klhl31 knockout lines at home and abroad. In order to identify the biological function of Klhl31 at the whole level of zebrafish, we used TALEN targeting technique to establish the Klhl31 gene knockout line of zebrafish. The results of restriction endonuclease analysis showed that the embryo injected with TALEN expression plasmid had a gene mutation at the restriction site, which destroyed the restriction site and led to the positive result of restriction endonuclease digestion, which proved the effectiveness of TALEN targeting. During the screening of F _ 0 mutant, the positive results were also detected by enzyme digestion. These results indicated that the Klhl31 gene of zebrafish was successfully knocked out by TALEN technique and the chimeric zebrafish with Klhl31 gene knockout was obtained.
【作者单位】：湖南师范大学生命科学学院蛋白质化学及鱼类发育生物学教育部重点实验室心脏发育研究中心;
【基金】：国家自然科学基金(81170229,81370451,31472060,81170088) 湖南省研究生科研创新基金(CX2011B195) 湖南省生物发育工程及新产品研发协同创新中心(2013-448-6)
【分类号】：Q78

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