电穿孔转染制备绵羊诱导多潜能干细胞条件的优化
Optimization of Inducing Pluripotent Stem Cells by Electroporation in Sheep

为优化获得绵羊诱导性多潜能干细胞（iPS cell）的诱导条件，通过4-D电转染仪，将含有人Oct4、Sox2及Klf4 、c-Myc及lin28基因的3个质粒共转染绵羊成纤维细胞，从EH-100、EN-150、CZ-167、CA-137电转程序筛选出适合绵羊成纤维细胞的电转染程序；探讨不同细胞密度、质粒质量浓度、质粒质量浓度比及维生素C对诱导性多潜能干细胞克隆形成的影响。结果表明，采用EH-100电转程序，在细胞密度为5×106 mL-1、质粒质量浓度比为1∶1∶1、质粒总质量浓度为0.1 mg·L-1、培养液中添加维生素C的质量浓度为0.05 mg·L-1时，AP染色阳性（AP+）克隆形成的效率可达到14.0%。说明，优化后的电转染方法和培养方案可有效提高绵羊诱导性多潜能干细胞的制备效率。
The aim of the experiment is to optimize and obtain the induction condition of induced pluripotent stem cells (iPS cell) in sheep, three recombinant plasmids which have the human gene of Oct4, Sox2and Klf4, c-Myc and lin28 was transfected into the sheep fibroblasts cell by 4-D electroporation.The suitable procedure for sheep fibroblasts cell was screened from EH-100, EN-150, CZ-167, CA-137.The impact on iPS cell colony formation was studied under different conditions, such as different cell density, plasmid mass concentration, plasmid proportion and culture medium with or without vitamin C.The Results indicated that the EH-100, the cell density of 5×106 mL-1, the three plasmids ratio of 1∶1∶1 , plasmid mass concentration of 0.1 mg·L-1 and the culture with 0.05 mg·L-1 vitamin C were the suitable programs, and the percentage of AP positive colonies reached to 14.0% in this program.Optimized electrical transfection methods and cultural programs can improve effectively the preparation efficiency of iPS cells in sheep.