Abstract：The objective of this study was to find a more efficient transfection method between a new transfection method and the traditional transfection methods. Three types of bovine donor cells (fetal fibroblast cells, fetal oviduct epithelial cells and cumulus granulosa cells) that commonly used for nuclear transfer were transfected with plasmid DNA of green fluorescent protein (GFP) gene using the nucleofection, electroporation and lipofection. We firstly determined optimal transfection parameters for nucleofection and electroporation, then transfection experiments were carried out to compare efficiency of optimized nucleofection and electroporation with lipofection under identical conditions, and green fluorescence ratio and cell survival were assessed 48 h after transfection. The results showed that the optimization program of nucleofection for fetal fibroblasts, fetal oviduct epithelial cells and cumulus granulosa cells were V013, T023 and U023; In electroporation, when the electric field was 90 V/mm, pulse duration was 10 ms, fetal fibroblasts and cumulus granulosa cells got the highest efficiency, while parameters of 80 V/mm and 5 ms were good for fetal oviduct epithelial cells; Finally, after transfection comparision experiments, the nucleofection demonstrated the highest transfection efficiency of (98.78±0.30)%、(88.43±2.10)% and (71.31±0.77)% respectively in all three types of cells, were significantly higher than electroporation and lipofection; and lipofection yielded the highest survival rate, electroporation got the lowest. The results demonstrate that nucleofection can be a more efficient alternative to produce transgenic cattle when combining with somatic cell cloning technology.
