Time course of fluorescence intensity and protein expression in HeLa cells stably transfected with hrGFP

The green fluorescent protein (GFP), obtained from Aequorea victoria, is widely used in transfection experiments to study the dynamics and characteristics of gene expression in a non-invasive manner. However, generation of cell lines displaying long-term expression of the GFP protein has repeatedly been reported unsuccessful. In the present study a different marker protein, humanized GFP (hrGFP) obtained from a sea pansy, Renilla reniformis, was used for transfection, and the time course of protein expression was studied by live cell fluorescence microscopy, PCR technology, and immunoblotting. HeLa cells were transfected with a plasmid, phrGFP-IRES-Neo containing the neomycin cassette and the hrGFP gene together with an internal ribosome entry site (IRES) sequence under the control of a CMV promotor. Cells were selected for three weeks after transfection using G418. During this time, hrGFP fluorescence was highest around day 4 and decreased thereafter eventually disappearing until the end of the selection period. In parallel, the proportion of cells staining positive for propidium iodide (i.e. dead cells) increased steadily. Three weeks after transfection non-fluorescent clones were present throughout. A couple of clones were subcultured for another week and used for further analyses. PCR methodology revealed the insertion of the hrGFP into the HeLa genome and the presence of mRNA species coding for hrGFP following reverse transcription. However, immunoblot analysis failed to show expression of the protein. These observations suggest a down-regulation of hrGFP expression at both transcriptional and post-transcriptional levels similar to results previously obtained with GFP. Thus, use of hrGFP does not appear to offer an advantage over GFP in transfection experiments aiming at permanent expression of the marker protein.