【摘要】 本研究以不同培养代数(P)水牛脂肪干细胞(buffalo adipose-derived stem cells,BASC)和成纤维细胞(buffalo fetal fibroblasts,BFFs)为研究对象,采用电穿孔法对其进行转染,比较转染后细胞的相对存活率和转染效率,为生产高效转基因供体细胞奠定基础。以绿色荧光蛋白(EGFP)为报告基因,电转染48 h后观察荧光表达,收集细胞采用血细胞计数法和流式细胞仪分析细胞相对存活率和转染效率。结果表明:电转后的BASC和BFF表达EGFP,BASC相对存活率低于BFF,P10之前BFF存活率更稳定;BASC和BFF转染效率随培养代数增加出现不同程度的降低,P10之前BFF转染效率更稳定。电转技术可用于转染BASC和BFF,并获得较高的转染效率;使用体外培养早期代数的BASC(P8之前)和BFF(P10之前)可以获得更稳定转染效率的转基因细胞。更多还原

【Abstract】 Different passages of buffalo adipose-derived stem cells(BASC) and buffalo fetal fibroblasts(BFF) were used to compare the relative viability and transfection efficiency after electroporation.This study will provide a good foundation to produce high efficient genetically modified donor cells for the transgenic clone buffalo research.Fluorescent microscope was used after 48 h to detect EGFP expression in transfect BASC and BFF.The relative viability was analyzed by blood cell count method,before the cell relative viability and transfection efficiency were analyzed by flow cytometry.The results showed that the BASC and BFF expressed the EGFP after electroporation.The cell relative viability of BASC were slightly lower than BFF(BASC:60%-70%,BFF:70%-80%).The transfection efficiency of BASC and BFFs reduced both with more passage,However,BFF showed a stable transfection efficiency before P10.Electrotransfection technology can be used in transgenic research of BASC and BFF,the early passages of BASC(before P8) and BFFs(before P10) are more suitable for electroporation with more stable transfection efficiency.更多还原