Effect of transfection manipulations on mouse cell cycle progression

Abstract

BalB/C-3T3 mouse fibroblasts and a temperature-sensitive derivative, ts 2e, were transfected by the calcium phosphate-dimethyl sulphoxide procedure to examine the effect of this manipulation on cell cycle progression. Cells were synchronized by growth to confluence in the presence of [2-14C]thymidine to generally label cellular DNA, and then subcultured from the G0 state. Plasmid pSV3-neo or pSV2-neo DNA was added to cells at 24 h post-plating, at peak Sphase. At designated intervals prior to, during, and after the transfection procedure, cells were labelled with [methyl-H]thymidine for 1 h to monitor nascent DNA synthesis and thereby assess cell cycle position. In all experiments performed, irrespective of the time of DNA addition, the transfection manipulations resulted in a reproducible, transient interruption of cell cycle progression, of about 5 h, and manifested as a delay in movement across the subsequent G1–S interface. Thereafter, the cycle resumed normally. The results indicated that the temporal sequence of the cell duplication cycle is altered when cells are exposed to exogenous DNA:Ca3 (PO4)2.Key words: transfection, cell cycle progression.