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利用CRISPR/Cas9技术构建tnnt2a基因突变斑马鱼及表型分析

刘恋, 张冉冉, 杨倩, 王旭, 桂永浩

复旦大学附属儿科医院心内科，上海 201102；复旦大学基础医学院生物化学与分子生物学系，上海 200032

摘要

心肌肌钙蛋白T (cardiac troponin T, cTnT)作为心肌纤维细肌丝的结构蛋白，参与心脏兴奋收缩耦联过程。tnnt2a为斑马鱼cTnT基因的亚型。本研究利用CRISPR/Cas9技术成功构建了可稳定遗传tnnt2a缺失突变斑马鱼。对tnnt2a缺失突变体进行表型分析，结果显示tnnt2a+/−斑马鱼心脏无明显异常，而tnnt2a−/−斑马鱼心脏在胚胎发育早期具有显著的突变表型，表现为心脏无搏动、心房和心室增大、心包积液，并于受精后6~7天死亡。运用real time RT-PCR检测tnnt2a、actc1a、tpm4a、myl7和vmhc基因在转录水平的表达情况，结果显示tnnt2a−/−(Δ2)斑马鱼tnnt2a基因在发育的各时间点表达水平均显著下降；而actc1a、tpm4a、myl7和vmhc基因在突变体胚胎发育早期表达增高，晚期则表达下降。进一步对心脏组织进行电镜观察和免疫荧光染色，结果显示tnnt2a−/−(Δ2)斑马鱼心脏无正常粗、细肌丝结构，同时F-actin与Tpm4的共染色也展示了心肌细胞肌丝结构的异常。本研究利用CRISPR/Cas9构建的tnnt2a突变斑马鱼表现出了心腔扩张，停搏及粗、细肌丝形成障碍等与临床扩张型心肌病相似的表型，因此可为TNNT2缺陷相关心肌病的研究提供很好的工具。

关键词： tnnt2a; CRISPR/Cas9 ; 斑马鱼

分类号：Q78

Generation of tnnt2a knock-out zebrafish via CRISPR/Cas9 and phenotypic analysis

LIU Lian, ZHANG Ran-Ran, YANG Qian, WANG Xu, GUI Yong-Hao

Department of Cardiology, Children’s Hospital of Fudan University, Shanghai 201102, China； Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Shanghai 200032, China

Abstract

Cardiac troponin T (cTnT) serves as a structural protein of myocardial fiber, and participates in heart excitation-contraction coupling process. Here, we generated tnnt2a (cTnT-coding gene) deletion mutant zebrafish via CRISPR/Cas9 technique, and performed phenotypic analysis of the identified tnnt2a mutants. We observed that there was no significant difference between heterozygous mutant and wild type zebrafish, and the homozygous mutants displayed significant malformations in heart, including cardiac arrest, atrium and ventricle enlargement, pericardium effusion, and the individuals usually died before 7 day post fertilization (dpf). We further analyzed the expression alternations of heart sarcomere genes (tnnt2a, actc1a, tpm4a, myl7, vmhc) at transcriptional level in tnnt2a−/−(Δ2) zebrafish by performing real time RT-PCR, and found that the RNA expression level of tnnt2a in tnnt2a−/− zebrafish decreased constantly at each time point of developmental stages, and actc1a, tpm4a, myl7 and vmhc all showed higher expressions at early developmental stages and lower expressions at late developmental stages, in comparison with those of wild type zebrafish. Lastly, electron microscopy on cardiac tissues suggested that there were significant changes of the thick or thin filament structures in tnnt2a−/−(Δ2) zebrafish, which was further confirmed by F-actin and Tpm4 immunofluorescence staining. The tnnt2a−/− zebrafish generated by CRISPR/Cas9 bears the most common symptoms of patients with dilated cardiomyopathy, and therefore can be used as a tool to study TNNT2-deficiency related cardiomyopathy.

Key words: tnnt2a; CRISPR/Cas9 ; zebrafish

收稿日期：2016-12-31　　录用日期：2017-05-02

通讯作者：桂永浩　　E-mail: yhgui@fudan.edu.cn

引用本文：

刘恋, 张冉冉, 杨倩, 王旭, 桂永浩. 利用CRISPR/Cas9技术构建tnnt2a基因突变斑马鱼及表型分析[J]. 生理学报 2017; 69 (3): 267-275.

LIU Lian, ZHANG Ran-Ran, YANG Qian, WANG Xu, GUI Yong-Hao. Generation of tnnt2a knock-out zebrafish via CRISPR/Cas9 and phenotypic analysis. Acta Physiol Sin 2017; 69 (3): 267-275 (in Chinese with English abstract).