猪早期胚胎发育中CDX2和OCT4基因功能及互作模式的研究

Pig is important farm animal and potentially useful in human disease model. However, deficiency of fundamental research in porcine embryonic development and related mechanism severely lagged pace of the adoption of pig as a model in human disease research. Even it was clear that CDX2 and OCT4 are the essential regulating genes during the formation of trophectoderm and inner cell mass in mouse blastocyst, unfortunately, the related knowledge and research in other large animal models is lacking. Up to data, preliminary results indicated that the expression patterns of CDX2 and OCT4 in embryos of livestock including pig, cattle and goat were different from the pattern in mouse embryo. Thus, in this study, we aimed to investigate OCT4 and CDX2 expression pattern, function and their interacting mechanism in early stage porcine embryos. In this work, porcine CDX2 and OCT4 gene will be characterized and cloned, and there expression pattern would be systematically traced by the method of immunofluorescence and in situ hybridization. Furthermore, through the RNA interference and over-expression in porcine zygote or one blastomere of 2 cell stage embryo, we would investigate their functions in embryonic development and first lineage segregation. Moreover, for further understanding the mechanism of OCT4 and CDX2 interaction, the protein-protein, and DNA-protein interaction would be investigated by the method of immunoprecipitation and chromatin immunoprecipitation. It is firmly convinced that through all these systematic research, as what we expected, not only the function and molecular mechanism of Oct4 and Cdx2 in early porcine embryonic development and first lineage segregation would be depicted clearly, but also a brand new insight and methodology in this area would be established, which will certainly promote the researches on pig as a human disease model.

猪是重要的肉用家畜和良好的潜在医学模型动物。但对猪早期胚胎发育的解析远不如小鼠清晰，这已构成猪在医学模型方面发展的瓶颈。CDX2和OCT4是小鼠囊胚滋养层和内细胞团形成关键调控基因，但在其他物种相关研究非常有限。现有研究表明猪、牛和羊等家畜胚胎CDX2和OCT4表达模式与小鼠并不一致。本课题将针对CDX2和OCT4在猪早期胚胎发育过程中的表达、胚层分化中的功能以及它们间相互作用机制进行研究。将分别钓取猪CDX2 和OCT4全基因序列，采用免疫荧光和原位杂交技术跟踪二者在猪早期胚胎的时空表达，通过过表达和敲低技术研究其对胚胎发育的影响，以单卵裂球过表达/敲低技术结合免疫沉淀和染色体免疫共沉淀来探明其胚层分化功能及相互作用机制。最终全面清晰地阐明CDX2和OCT4在猪胚胎第一次胚层分化中的功能及作用机制。该研究将为系统解析猪早期胚胎发育奠定理论和方法学基础，并将直接促进猪作为医学模型相关研究。

猪是重要的肉用家畜和良好的潜在医学模型动物。但对猪早期胚胎发育的解析还远不如小鼠清晰，这已构成猪在医学模型方面发展的瓶颈。CDX2和OCT4是小鼠囊胚滋养层和内细胞团分化的关键基因，但在其他物种中相关研究非常有限。现有结果表明猪、牛和羊等家畜胚胎CDX2和OCT4表达模式与小鼠不一致。本项目在前期研究基础上，通过获取猪CDX2和OCT4基因信息获得体外过表达载体和干扰片段。进一步在猪早期胚胎中研究CDX2在猪滋养层细胞分化中的功能以及其对猪卵裂球命运的影响。最后在分子水平上明确CDX2和OCT4在猪早期胚胎中的相互作用机制。本研究的结果丰富了对哺乳动物早期胚胎发育胚层分化机理的认知，为进一步阐明猪早期胚胎发育滋养层与内细胞团分化机理奠定了基础。