| 利用CRISPR/Cas9构建斑马鱼tbx20基因突变体及其功能分析 |
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| 引用本文： | 朱哲,胡沛男,李伟明,祖尧.利用CRISPR/Cas9构建斑马鱼tbx20基因突变体及其功能分析[J].上海海洋大学学报,2019,28(1):1-9. |
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| 作者姓名： | 朱哲 胡沛男 李伟明 祖尧 |
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| 作者单位： | 上海海洋大学科技部海洋生物科学国际联合研究中心;上海海洋大学水产种质资源发掘与利用教育部重点实验室;上海海洋大学水产科学国家级实验教学示范中心 |
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| 基金项目： | 国家自然科学基金（31501166）；上海市教育委员会晨光计划项目（14CG49） |
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| 摘 要： | 转录因子TBX20在脊椎动物心脏的腔室发育和维护中起至关重要的作用。利用CRISPR/Cas9系统成功制备了斑马鱼tbx20突变鱼系,T7E1检测结果显示F_0敲除效率平均为42. 1%,测序分析F_1中突变种质遗传效率为36. 7%。F_2突变体中观察到心脏突变表型:48 hpf,心包腔肿大,静脉窦瘀血,环化异常,心脏结构变形; 3 dpf,心脏畸形拉伸成线状结构。原位杂交和qRT-PCR结果显示,突变体中vmhc表达上调,amhc和myl7表达下调。成功制备了斑马鱼tbx20突变体,结果表明tbx20纯合突变体心脏畸形,环化受到影响,为深入探究tbx20在早期心脏腔室分化过程中的作用奠定了基础。
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| 关 键 词： | tbx20 斑马鱼 心脏发育 腔室分化 CRISPR/Cas9 |
| 收稿时间： | 2018/5/8 0:00:00 |
| 修稿时间： | 2018/11/20 0:00:00 |
Zebrafish tbx20 gene knockout using CRISPR/Cas9 system and its related functions analysis |
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| ZHU Zhe,HU Peinan,LI Weiming and ZU Yao.Zebrafish tbx20 gene knockout using CRISPR/Cas9 system and its related functions analysis[J].Journal of Shanghai Ocean University,2019,28(1):1-9. |
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| Authors: | ZHU Zhe HU Peinan LI Weiming and ZU Yao |
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| Institution: | International Research Center for Marine Biosciences, Ministry of Science and Technology, Shanghai Ocean University, Shanghai 201306, China;Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306, China;National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China,International Research Center for Marine Biosciences, Ministry of Science and Technology, Shanghai Ocean University, Shanghai 201306, China;Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306, China;National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China,International Research Center for Marine Biosciences, Ministry of Science and Technology, Shanghai Ocean University, Shanghai 201306, China and International Research Center for Marine Biosciences, Ministry of Science and Technology, Shanghai Ocean University, Shanghai 201306, China;Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306, China;National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China |
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| Abstract: | The tbx20 mutated zebrafish line is successfully established by using CRISPR/Cas9 system. The tbx20 mutagenesis efficiency is about 42.1% which was evaluated by T7E1 assay in F0. Sequencing analysis estimated the germline transmission efficiency of tbx20 mutagenesis is 36.7% in F1. The tbx20 homozygote mutant showed obvious cardiac phenotypes:the tbx20 mutant displayed pericardial oedema, sinus congestion, looping abnormal and the cardiac morphology deformation at 48 hpf; all the phenotypes became severer at 3 dpf and tbx20 mutant showed linear heart. In situ hybridization and realtime fluorescence quantitative PCR results revealed that the expression of vmhc was upregulated, amhc and myl7 in the tbx20 mutants were downregulated. The tbx20 mutated zebrafish line was successfully produced and was confirmed tbx20 affected heart looping, which established a foundation for future studies on the function of tbx20 gene in early cardiac chamber differentiation. |
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| Keywords: | tbx20 zebrafish cardiac development chamber differentiation CRISPR/Cas9 |
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