| Heart is one of the earliest developmental and functional organs of vertebrate, and its development process is regulated by a lot of genes. The abnormal expression of these genes may result in different cardiac defects. In our country, there are about10million patients suffer from congenital heart disease and this kind of disease is known as the number one killer of human. Therefore, researches on the regulatory genes of cardiac development help to better understand the pathogenesis of heart disease and is of great significance for the prevention and treatment of the disease. Zebraflsh, as a kind of ideal animal models, has advantages such as individual small, easy to breed, rapid development, strong reproductive capacity, in vitro fertilization, embryo development in vitro and transparent, and its genome similarity compared with human is as much as87%, so using zebrafish as a vertebrate developmental biology model to study the mechanism of gene regulation of cardiac development has a big superiority.Our previous studies showed that the zebrafish gene HPRG1may be involved in the early morphogenesis of the heart and affected the BMP signaling pathways that plays an important role in heart development by interacting with Smad proteins. In this paper, zebrafish was used as a model for further study of the functions of HPRG1in vertebrate heart development. This paper first studied in HPRG1gene expression pattern in zebrafish embryonic development through the use of whole embryo in situ hybridization. The experimental results showed that the HPRG1gene expressed at the early stages of embryonic development, and specifically expressed in the heart tissues at later stages. These results are coincident with the previous results of embryonic immunofluorescence experiment. Moreover, this paper uses To12transposon system to generate a series of transgenic zebrafish lines related to zebrafish heart candidate gene HPRG1, including:Tg: pTol2(cmlc2: HPRGl-EGFP), Tg: PTol2(HPRG1(3kb):EGFP), Tg: pTol2(HPRG1(2kb):EGFP), PTol2(HPRG1(1kb):EGFP). The results of phenotypic analysis of pTol2(cmlc2:HPRG1-EGFP) line shows that overexpression of HPRG1in heart leads to many cardiac defects, including larger pericardial cavity, abnormal cardiac cyclization, smaller ventricle and larger atrium.The subsequent M-mode physiological analysis further proved that the HPRG1gene played an important role in normal development of the heart. The results of phenotypic analysis of the pTol2(HPRG1(3kb): EGFP) line showed that3kb promoter restricted HPRGl gene expressed in the heart tissues specifically, and its expression pattern is coincident with the previous result of in situ hybridization. In addition, this paper also generated a pTol2(cmlc2: CRE-IRES-EGFP) line, which specifically expressed Cre recombinant enzyme in heart and has no significant effect on the normal development of the hear.Thus, the successful generation of this transgenic zebrafish line laided foundation for the study of the function of heart development candidate genes in heart development and related heart diseases.In this paper, CRISPR/Cas9targeting technology was used to knockout zebrafish gene HPRG1. Through the website analysis, we identified the most appropriate target site of the HPRG1gene and constructed the left and right half sites CRISPR expression plasmids, which was then transcribed into RNA and microinjected into zebrafish one cell phase embryos mixed with Cas9endonuclease mRNA. With using the extracted genome of24hpf embryos as template for PCR amplification and subsequent enzyme digestion,we confirmed that the expression vectors can induce gene mutation effectively.The generation of stable knockout model of zebrafish gene HPRG1is underway. |
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