| With the rapid development of the society and growing issue of environmental pollution, infertility is increasing. According to the World Health Organization survey, infertility is becoming the third largest disease after cancer and cardiovascular disease in the21st century, one of the main reason is no effective treatment for uterine hypoplasia. HPS is an autosomal recessive genetic disease, the main clinical symptoms of which is fibrosis, bleeding, colitis. Pearl (pe)phenotype is a typical HPS Ⅱ animal models by the mutation of HPS related loci encode protein, AP3B1. There are no report about fertility ability in HPS II patients and pe mice.We have preliminary data demonstrating that the litter size of pe female mouse is remarkably reduced and the the chances of pregnancy is slightly lower.To investigate the underlying mechanism of the reduced fertility, we examined its reproductive organs at several levels in pe female mouse:1) in tissue level, uterine dysplasia can be detected from one-month-old mouse, resulting in a "Immature uterus’ in adult; there were no obvious abnormalities detected in pe female ovaries, but the number of eggs in super-ovulation is significantly reduced.2) in estrogen level, no significant changes can be detected in pe female mouse.3) in molecular level, Hoxa10, Hoxa11, Wnt5a, the critical gene for uterime development is down regulated; the expression level of β-catenin, a key gene in Wnt pathway regulating uterine development, increased in cytoplasm and decreased in cell membrane. We proposed that AP3B1mutations affect transport β-catenin in the barrier, leading to its accumulation in the cytoplasm. Simultaneously, we supposed that the disruption of the AP-3pathway is possible to induce abnormal cellular location of PCP-2, which in turn remove the inhibition of the beta-catenin signaling and further lead to disregulation of Wnt and Hox signaling pathway.In summary, pe female mouse with significant uterine hypoplasia characteristics is a good animal model for the uterine hypoplasia study. Thus, a better understanding of the mechanisms regulating uterine hypoplasia in pe mouse will improve insight into infertility study. Genome editing is a technology for genome modification of specific sites. It has promised applications in the study of gene function, gene repair and cell replacement therapy. The CRISPR-Cas9system has been developed as an efficient gene editing technology in cells and organisms. The purpose of this part of the thesis is to build a mouse zygote microinjection platform and explore the application of CRISPR-Cas9systems in the gene knockout at mice.The mouse zygote microinjection platform consists of four parts, including the collection of fertilized eggs, fertilized eggs treatment, microinjection of fertilized eggs and two-cell stage embryo transfer. We optimized a set of experimental procedures for the above four steps through repeated experiments, including the best of hyaluronidase concentration (0.3mg/ml), the best fertilized egg collection time (13:00pm), the most appropriate pseudopregnant female mouse strain for embryo transfer (CD1). We have obtain many new-born mice through embryo transfer, indicating that the microinjection platform is completed.We try to knockout the epididymis-specific gene, including the Teddml and Serpinalf, by CRISPR-CAS9microinjection. Due to time constraints, we completed the design of these targeted genes, and the constructions of gRNA and Cas9RNA are underway. We believe that the CRISPR-Cas9systems will be fully developed in the field of genetic modification in future, and promote the study of gene function. |
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