Study On Gata4 Gene Knockout Model Of Zebrafish Congenital Heart Disease By TALEN Technique

Objective The congenital heart disease of human was related to the mutation of gata4 gene, contract the animal model by gene knock out technique for the research of CHD’s pathogenic mechanism and gene therapy. Nowadays transcription activator like effectors nucleases (TALEN) is matured and most widely used of gene target modification technique, it includes two main domain:specific nucleases-binding domain and endonuclease enzyme cutting domain. Artificially contract the TALEN vectors that targeting the first exon of gata4 gene, it can induce double-strand break of DNA at the specific site and trigger the cell’s own DNA repair mechanisms of NHJE, then, it will introduce the gene mutation, knock out and knock in, thus realizing targeting targeted genome editing, and established the gata4 gene knock out zebrafish model.Methods Bioinformatics methods were used to analyze the DNA coding sequence of zebrafish gata4 gene, design and select the appropriate targeting sites on websites, then we design a pair of TALEN that target the first exon of gata4 gene. "UA" method was used to assemble specific TALE binding domain, bacteria-PCR and sequencing were used to confirm whether TALE is assembled correctly. The TALE binding domain was cloning into the expression vectors of pCS2-PEAS and pCS2-PERR, then we obtain TALENs of the left and right half-site, bacteria-PCR, restriction enzyme digestion and sequencing were used to confirm whether vectors are correct. The linearized DNA vectors were transcribed into mRNAs using SP6 mMESSAGE mMACHINE Kit in vitro, then microinjected TALEN mRNAs into zebrafish embryos, the activity of TALEN in vivo was detected by enzyme digestion method. After the injected zebrafish sexual maturity, the mutations of zebrafish were screening through PCR and enzyme digesting, TA-clone sequencing to confirm the genotype of mutantions and obtain the model of gata4 knock-out zebrafish.Results We successfully constructed correct targeting vectors by enzyme digesting and sequencing, which the efficiency was 35.18%, then we screened the mutant zebrafish and confirmed the different types of mutation.Conclusions The gata4 knock-out zebrafish model was successfully established, it can provide a good animal model for further research of congenital heart disease.