Studies On The Factors Influencing Establishment Of Cell Line Porcine Embryonic Fibroblasts And Cationic Liposome Mediated Transfection

In vitro culture system of procine embryonic fibroblasts(PEF)and preferential transfection method was investigated in this study.cell line of porcine embryonic fibroblasts(PEF) was established by embryo of 26th WZSP and was subcultured to 27th passages successfully. moreover,there was 48% of cells transfected positively by use of optimum condition of transfection.According to the behest of cell line,the present study performed a biological study,the results as follows: 1.PEF was consist with morphological character of classic fibroblast, and could be purified for their different sensibility to trypsin. 2.The growth curve of PEF did not show significant difference among 2th,5th,10th passages,and went through lag phase,log phase,plateau phase. 3.PEF were frozen in DMEM with serum and adding 10% DMSO and 10% glycerol respectively, The survival rates of PEF were 76.93%,81.74% individually, they are both can meet the needs of fibroblast passage and log-term preservation. 4.Karyotype analysis indicated that PEF had 2n=38 chromesomes, this culture system was suitable and safe to PEF, and keep relatively stable karyotype. 5.pEGFP-N3 could efficiently transfected the PEF by Fugene-6.The present study optimize several factors influence transfection efficiency;confluence rate of PEF(50%~60%), proportion of plasmid DNA(1.0μg)/liposome(3.0μl),transfection time(8 h),concentration of G418(800μg/ml), and transfection efficiency can gradually increased to 48%.