Liposome-mediated Gene Transfection Of Mouse Somatic Cells

For liposome-mediated gene tranfer system, the main problem is the lower transfection efficiency, which inhibits its use more popularly. In our experiments, we used GFP gene as reporter gene to study how the factors affected the transfection efficiency and studied what effects the liposome method took on mouse somatic cells in order to increase its transfection efficiency.First, we studied the effects of the amount of liposome and plasmid, exposure time of cells to the liposome-plasmid complexes, number of cell passages and cell types on GFP gene transfection of mouse somatic cells. Second, under the optimal conditions, we compared the effects of the number (from primary to 15) of passages and cell types on the transfection efficiency. Third, we selected the better cell cycle synchronization methods, and with the better methods we studied the effects of cell cycle stages on liposome tranfection efficiency. In the whole, our results are summarized as follows:1. The maximal GFP transgene expression (30.7%) was achieved when mouse fetal fibroblast cells (MFFC) at 70%-90% confluence of passage 3 were exposed for 6 h to the complexes of 4μg liposome (LipofectAMINE) and 0.3ug plasmid (pEGFP-N1) per wellof a 24-well plate.2. Under these conditions, we compared the effect of the number (from primary to 15) of passages on the transfection efficiency of MFFC. The transfection efficiency of MFFC was 10.0%, 28.9%, 28.7% and 7.2% at primary, 3, 6 and 15 passages, respectively, indicating that the transfection efficiency was maximal and decreased with passaging.3. When MFFC, mouse oviductal epithelial cells (MOEC) and mouse granulosa cells (MGC) were transfected at passage 3, the transfection efficiency was 27.8%, 13.7% and 14.2%, respectively, under the described transfection conditions. So different cell types had different transfection efficiency. Moreover, when the cell cycle stages of different cell passages and types at were examined, we found transfection increased with the augment of percentages of cells at G2/M phase.4. The best synchronization results were obtained when MFFC were treated 5 hours at 5℃, and the cold shock led to the highest G0/G1 phase accumulation (74.1%) and the lowest apoptosis (3.5%). Also, Ros induced the G0/G1 phase accumulation. The highest G0/G1 phase and the lowest apoptosis were 84.2% and 9.4%, respectively. And the best results were achieved with culture medium containing 10 μM Ros for 24h.Because cold shock induced lower cell cycle synchronization than Ros and Ros led more cells to apoptosis than cold shock, we used cold shock commoned with Ros for better synchronization. When cells were cooled to 5℃ for 5h and then returned to theculture medium containing 10 uM Ros for 12h at 37 °C (LTR2), the percentage of GO/Gl phase and the apoptosis were 83.6% and 3.6%, repectively. And serum starvation for 48h led to 79.7% of GO/Gl phase and the apotosis was 8.0%. Coichicine and nocodazole treatment induced 34.7% and 32.4% G2/M phase accumulation, and led to 25.3% and 21.9% apoptosis, respectively. From the above results, the better synchronization methods included cold shock for 5h, 10 uM Ros for 24h at 37 °C, LTR2 and serum starvation for 48h. When cells were released from the four methods, samples were harvested at 4h intervals and analyzed cell cycle status. Serum starvation did not get higher percentage of S phase and G2/M phase, while the other three methods did get higher percentage of S phase and G2/M phase.5. MFFC were transfected at GO/Gl, S and G2/M phase, and the results indicated that the cell and clone number of expressing GFP were maximal at G2/M phase. In conclusion, G2/M phase considerately enhanced liposome-mediated transfection efficiency.6. The results showed that the cells were not normal and healthy in morphology, and there were more cells going into apoptosis sighnificantly (PO.05) and more cells arrested in GO/Gl phase after transfection. But their karyotype did not differ sighnificantly (P>0.05) after transfection.hi conclusion, higher transfection efficiency could be achieved with cold shock, Ros treatment and LTR2 synchronization methods than with other methods. And LTR2 led to the highest transfection efficiency.