Zebrafish tbx20 gene knockout using CRISPR/Cas9 system and its related functions analysis

doi:10.12024/jsou.20180502308

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Zebrafish tbx20 gene knockout using CRISPR/Cas9 system and its related functions analysis
DOI:
                        10.12024/jsou.20180502308
                    
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Affiliation:International Research Center for Marine Biosciences,Ministry of Science and Technology,Shanghai Ocean University;Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,Ministry of Education,Shanghai Ocean University;National Demonstration Center for Experimental Fisheries Science Education,Shanghai Ocean University,International Research Center for Marine Biosciences,Ministry of Science and Technology,Shanghai Ocean University;Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,Ministry of Education,Shanghai Ocean University;National Demonstration Center for Experimental Fisheries Science Education,Shanghai Ocean University,International Research Center for Marine Biosciences,Ministry of Science and Technology,Shanghai Ocean University,International Research Center for Marine Biosciences,Ministry of Science and Technology,Shanghai Ocean University;Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,Ministry of Education,Shanghai Ocean University;National Demonstration Center for Experimental Fisheries Science Education,Shanghai Ocean University
Clc Number:S917
Fund Project:【Supported by the National Natural Science Foundation of China (No. 31501166), Chenguang Program of the Shanghai Municipal Education Commission (14CG49)】

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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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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.
ZHU Zhe, HU Peinan, LI Weiming, 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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Received:May 08,2018
Revised:November 20,2018
Adopted:November 21,2018
Online: January 21,2019
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