【摘要】 目的人工构建TALEN靶向敲除载体敲除gata4基因,建立斑马鱼先天性心脏病动物模型。方法采用单元组装法构建靶向敲除gata4基因的载体,将其体外转录成TALEN mRNAs,通过显微注射的方式注入单细胞期受精卵中,胚胎检测TALEN的敲除效率,再通过剪尾、酶切、测序筛选发生基因突变的斑马鱼及突变的类型。结果酶切、测序结果证实成功构建出正确的gata4靶向敲除载体,注入斑马鱼受精卵中,发生基因敲除的效率为35.18%,通过剪尾、PCR、酶切检测成功地筛选出了发生基因突变的斑马鱼,测序结果显示出不同种类的gata4基因突变。结论成功构建gata4基因敲除的斑马鱼动物模型,为进一步研究人类gata4基因突变引起先天性心脏病的发病机制提供了良好的动物模型。更多还原

【Abstract】 Objective To establish a gata4 gene knockout zebrafish model of congenital heart disease,and construct transcription activator-like effector nuclease( TALEN) vectors targeting gata4 gene. Method We construct TALEN vectors targeting zabrafish gata4 gene using unit assembly method and the in vitro-transcribed TALEN mRNAs were microinjected into one-cell stage zebrafish embryos. The efficiency of TALEN was identified by injected embryos,and mutations of zebrafish were screened and confirmed the different types through PCR and enzyme digestion. Results We successfully constructed correct targeting vectors by enzyme digestion and sequencing,and the gene knockout efficiency was35. 18%. We screened the mutant zebrafish and confirmed different types of gata4 gene mutations. Conclusions A gata4 knockout zebrafish model is successfully established,it can provide a good animal model for further research of congenital heart diseases.更多还原