生产转基因五指山小型猪重构胚条件优化

Study on production of transgenic Wuzhishan miniature pigs embryos by somatic cell nuclear transfer

利用体细胞核移植技术是得到具有优良特征及抗病性五指山小型猪快速有效方法,因此将外源基因安全高效导入到供核细胞至关重要。利用不同转染试剂、转染方法对转染效率进行研究,确定G418筛选浓度。同时利用体细胞核移植技术生产转单体红色荧光蛋白重构胚。结果表明,使用U-023细胞核电转程序能有效介导质粒pCX-mRFP1转染猪耳成纤维细胞,转染率可达到83.33%。确定G418最佳筛选浓度为200μg.mL-1。利用转基因细胞作为核供体生产重构胚卵裂率是83.08%(221/266),囊胚率是11.65%(31/266)。研究结果为生产表达单体红色荧光蛋白五指山小型猪提供参考,从而为人类疾病发病机理研究以及生产人类疾病模型动物奠定了基础。

Production excellent characteristics and disease resistance of Wuzhishan miniature pig by somatic cell nuclear transfer is a fast and effective method, therefore exogenous gene into nuclear cell is very important. This study was performed to compared the transfection efficacy of several transfection reagent and methods, screening concentration of G418. Production expression of monomeric red fluorescent protein in transgenic Wuzhishan miniature pigs embryos by somatic cell nuclear transfer. Conclusion： The optimize transfection used an electroporation-based Lonza Nucleofector system, the rate of transfection was 83.33%. Screening concentration of G418 was 200 pg/ml, Donor cells transfected with pCX-mRFP1 gene were used in nuclear transefer, the overall developmental rates of transgenic SCNT embryos showed the normal cleavage rate was 83.08%, blastocyst formation rate was11.65%. The study therefore provides information for establish the system to get transgenic minipigs by SCNT, supplied the basis to study the pathologic mechanism of human disease and produce the animal model for human genetic disease.