Quantitative analysis of transient gene expression in mammalian cells using the green fluorescent protein.
The green fluorescent protein (Gfp) has been used as a reporter, along with flow cytometric analysis, to follow the dynamics of gene expression in transiently transfected mammalian cells. Gene transfer conditions for lipofection were optimized. The highest fraction of transfectants were obtained when lipid-DNA complexes were formed with 6 microliters lipid and 1 microgram DNA for chinese hamster ovary (CHO) cells and with 9 microliters lipid and 2 micrograms DNA for NIH/3T3 cells. Chinese hamster ovary cells were monitored for Gfp expression and growth for 6 days following transfection. An initial decrease in viability for 36 h was observed after which cell growth followed exponential kinetics with increasing viability. Intracellular accumulation of recombinant protein peaked at 24 h post-transfection and then decreased with first order kinetics at a rate comparable to the specific growth rate. It appears that dilution by growth accounts for the decrease of Gfp in the biomass. Immunofluorescent staining of Gfp and subsequent flow cytometric analysis of transfected cells revealed a linear correlation between the green fluorescence and immunofluorescence. This indicates that green fluorescence is a quantitative measure of intracellular Gfp in single cells in spite of the dynamics of post-translational modifications involved in the conversion of expressed protein into its fluorescent form. A structured model has been formulated to describe the observed kinetics of gene expression and fluorophore formation. The model accurately predicts experimental trends and suggests that the fraction of non-fluorescent Gfp is significant only during the initial period of gene expression.
Subramanian
S
S
Institute for Advanced Studies in Biological Process Technology, University of Minnesota, St. Paul 55108, USA.
Srienc
F
F
D016475:3T3 Cells; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D008322:Mammals / Q000235:genetics; D051379:Mice; D008957:Models, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D014162:Transfection
D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins