Effects of cell cycle status on the efficiency of liposome-mediated gene transfection in mouse fetal fibroblasts.
Methods for cell cycle synchronization of mouse fetal fibroblast cells (MFFCs) were first selected and optimized. When MFFCs were cooled at 5 C for different periods of time, the highest percentage of cells at the G0/G1 phase (75.4+/-2.9%), with 3.5+/-0.3% of apoptotic cells, was achieved after 5 h of treatment. Extended cooling increased the number of apoptotic cells significantly. When MFFCs were treated with different concentrations of roscovitine (ROS) for different periods of time, the highest percentage of G0/G1 cells (83.5+/-1.8%), with 9.2+/-0.6% apoptotic cells, was obtained after exposure to 10 microM ROS for 24 h. When the cells were cooled at 5 C for 5 h followed by incubation in 10 microM ROS for 12 h, 83.6+/-1.9% were synchronized at the G0/G1 stage, with 3.6% undergoing apoptosis. Cell cycle progression was then observed after release of the MFFCs from different synchronization blocks. The highest percentages of S and G2/M cells (81% and 75%) were achieved at 12 and 20 h, respectively, after release of the MFFCs from the cooling plus ROS treatment, and these percentages were significantly higher than those obtained after release from the cooling or ROS alone blocks. Finally, MFFCs were transfected with pEGFP-N1 plasmid at the peak of the G0/G1, S, and G2/M phases, respectively, after release from the different blocks and both the transient and stable transfection efficiencies were determined. The GFP gene expression was greatly enhanced when transfection was performed at the time when most cells were at the G2/M stage after release from cooling, ROS alone, and cooling plus ROS treatments. Statistical analysis revealed a close correlation between the rate of G2/M cells and the transient and stable GFP gene expression efficiencies. Together, the results indicated that (a) the best protocol for cell cycle synchronization of MFFCs was a 5-h cooling at 5 C followed by incubation in 10 microM ROS for 12 h which produced both a high rate of synchronization in the G0/G1 phase with acceptable apoptosis and a high rate of G2/M cells after release; and (b) that the cell cycle status had marked effects on the efficiency of liposome-mediated transfection in MFFCs, with the highest transfection efficiency obtained in cells at the G2/M stage.
Yu
Jian-Ning
JN
College of Animal Science and Veterinary Medicine, Shandong Agriculture University, China.
Ma
Suo-Feng
SF
Miao
De-Qiang
DQ
Tan
Xiu-Wen
XW
Liu
Xin-Yong
XY
Lu
Jin-Hua
JH
Tan
Jing-He
JH
D000818:Animals; D017209:Apoptosis; D002453:Cell Cycle; D002478:Cells, Cultured; D005333:Fetus / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D007399:Interphase; D008081:Liposomes; D051379:Mice; D011687:Purines / Q000494:pharmacology; D000077546:Roscovitine; D013696:Temperature; D014162:Transfection / Q000379:methods* / Q000592:standards
D008081:Liposomes; D011687:Purines; C485184:enhanced green fluorescent protein; D000077546:Roscovitine; D049452:Green Fluorescent Proteins