用于帕金森氏症研究的GFAP-DsRed转基因巴马小型猪模型构建

The pig is a good model for biomedical research and transgenic pig is an important big animal model for translational biomedical research on particular human diseases. Parkinson's disease is a progressive neurodegenerative disease and the research of it needs big animal models of which pathological process can be tracked. At present there is not any transgenic pig model for studying Parkinson's disease. In this project Guangxi Bama minipig and reproductive biotechnology are used. The GFAP-DsRed expression vector is constructed and transfected into Bama minipig somatic cells, and transgenic embryos are produced using somatic cell nuclear transfer technique and transferred into recipient pigs. A batch of transgenic Bama minipigs expressing DsRed specifically in central nervous system will be obtained and the second generation of GFAP-DsRed transgenic Bama minipig will also be obtained. The second generation of transgenic Bama minipig will be then used for studying Parkinson's disease by being treated with neurotoxicant to induce Parkinson's disease and a transgenic Bama minipig model for studying Parkinson's disease will be developed at the end of this project. The distinguishing feature of this transgenic Bama minipig model is that the degree of damage of central nervous system can be judged by tracking the expression status of DsRed in central nervous system thus provide a new tool for studying Parkinson's disease. This project will also enrich the technologies and theory in minipig reproduction and open new ways for protection and improvement of Guangxi local pig breeds with important variety and economic value.

猪是生物医学研究良好模型，转基因猪是人类特定疾病研究的转化生物医学重要大动物模型。帕金森氏症是一种渐进神经退行性变疾病，其研究需能对其病理进程进行示踪的大动物模型，目前国内外尚无用于帕金森氏症研究的转基因猪模型。本项目选用广西巴马小型猪为材料，利用繁殖生物技术，构建GFAP-DsRed表达载体，通过体细胞核移植生产转基因胚胎、进行胚胎移植，获得在中枢神经系统(CNS)特异表达红色荧光蛋白(DsRed)的转基因巴马小型猪，繁殖并选育出第二代GFAP-DsRed转基因巴马小型猪，利用神经毒素处理该第二代转基因巴马小型猪进行帕金森氏症研究，最终开发出用于帕金森氏症研究的转基因巴马小型猪模型。该模型特色在于通过追踪DsRed在CNS表达情况判断CNS受损害程度，为帕金森氏症研究提供新工具。本项目还将丰富小型猪繁殖学领域的技术和理论，同时为具有重要经济价值的广西地方猪品种的保种和品种改良开辟新途径。

本研究旨在构建由人胶质纤维酸性蛋白（Glial fibrillary acidic protein, GFAP）基因启动子控制在中枢神经系统的星形胶质细胞中特异表达DsRed的转基因广西巴马小型猪，从而为帕金森氏症研究提供一种大动物模型。本研究开展了以下工作：①广西巴马小型猪转基因克隆胚胎的制备；②通过提高移植胚胎数量来提高GFAP-DsRed转基因广西巴马小型猪的产出效率；③对GFAP-DsRed转基因广西巴马小型猪进行表型分析；④对GFAP-DsRed转基因在广西巴马小型猪的肝脏和胰腺星形细胞中是否可以特异性表达DsRed进行探索。经Xfect polymer转染制备的DsRed转基因广西巴马小型猪体细胞形态正常、活力良好，并且转基因操作不会影响广西巴马小型猪克隆胚胎的体外发育潜力，制备了DsRed转基因广西巴马小型猪克隆胚胎；通过显著提升移植胚胎数量的方法（从148.2±13.0枚提高至大于200枚）使得成功制备16头存活的GFAP-DsRed转基因广西巴马小型猪公猪，这些克隆猪均生长发育正常并经基因型鉴定为阳性转基因；对GFAP-DsRed转基因广西巴马小型猪进行表型分析，结果表明人GFAP基因启动子含有相匹配的基因元件，能够在猪大脑星形胶质细胞中驱动DsRed特异性表达；对GFAP-DsRed转基因在广西巴马小型猪的肝脏和胰腺星形细胞中是否可以特异性表达进行了探索，结果表明在GFAP-DsRed转基因广西巴马小型猪肝脏和胰腺星形细胞中能够特异性启动DsRed表达。我们成功构建了GFAP-DsRed转基因广西巴马小型猪，为帕金森氏症的大动物模型研究奠定基础。