Generation Of Zebrafish Mutant Lines Of The Heart Candidate Gene Hprg1 And A Preliminary Study On Its Function

Heart disease is regulated by multiple genes,researches on related genes of cardiac development is the breakthrough of human's challenge to overcome the disease.While we study gene biological function by knockout and knockin mostly.Three years ago,gene edit artifact CRISPR(Clustered regularly interspaced short palindromic repeats)is considered to be a revolutionary technology in the field of genetics research and as a"lightning" trend sweep the domestic and international scientific community.Zebrafish as a classic laboratory model are used in areas such as genetics and molecular biology research with its own superiority,and our laboratory has a perfect research platforms of model organism zebrafish.hprgl gene is one of the heart development candidate gene identified in our laboratory.There is no relevant reports at home and abroad to establish the gene mutant lines.In order to explore the biological function of hprgl in zebrafish,We used CRISPR/Cas9 technology to construct hprgl gene mutant lines in zebrafish.Firstly we analysis hprgl gene function domains with the websites.Then we design nine gRNA which respectively in four,five,and six exons of the gene.Then we co-inject into zebrafish embryos at one cell stage with hCas9 mRNA and gRNA are,we test validity of embryonic genome at 48hpf.The FO positive fishes with three months old were used for tails cutting for obtaining chimeric knockout fish.Then FO generation of positive sexual maturity chimeras were hybridized with wild type to get the F1 hybrids which were screened with similar methods.F1 hybrids which have the same mutations were crossed to get F2 generation homozygote.Now,we have established three knockout models which are 16bp,17bp deletion and 19bp insertion.We then evidence that the dead rate of mutant are higher than the wild-type.The research has laid a solid foundation for the further study of molecular biological function of hprgl gene from overall embryonic level and the regulatory mechanism of heart development,and provide new molecular target sites for clinical treatment.