激光生物学报, 2016, 25 (6): 546, 网络出版: 2017-01-23  

斑马鱼心脏发育候选基因Klhl31的TALEN打靶有效性分析

Targeting Effectiveness Analysis of Heart Developmental Candidate Gene Klhl31 with Gene Knockout Method TALEN
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, 湖南 长沙 410081
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 technology is a method of gene knockout orientation, which can solve the problem that is difficult for in vivo gene knockout in zebra fish at present. Klhl31 is one of human heart developmental candidate genes identified in our laboratory. So far there is no Klhl31 gene knockout strains in zebra fish at home and abroad. In order to identify the biological function of Klhl31 gene in zebra fish, we used TALEN technique to build Klhl31 gene knockout strains in zebra fish. Digestion analysis showed that mutational DNA band occurred at the cleavage site in those injected embryos with TALEN expression plasmids, suggesting that the restriction sites were destroyed, which demonstrated that TALENs technology was effective in this experiment. In the selection of F0 generation of mutants, the positive results were confirmed by enzyme digestion. These results suggested that the application of TALEN techniques to knockout Klhl31 gene was successful, and the chimerical Klhl31 knockout zebrafish was obtained.

蔡湾湾, 陈发, 万永奇, 吴秀山, 邓云, 袁婺洲. 斑马鱼心脏发育候选基因Klhl31的TALEN打靶有效性分析[J]. 激光生物学报, 2016, 25(6): 546. CAI Wanwan, CHEN Fa, WAN Yongqi, WU Xiushan, DENG Yun, YUAN Wuzhou. Targeting Effectiveness Analysis of Heart Developmental Candidate Gene Klhl31 with Gene Knockout Method TALEN[J]. Acta Laser Biology Sinica, 2016, 25(6): 546.

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