This is a photomicrograph of some human embryonic kidney cells (HEK 293 cells) that I grew. Notice the three cells near the center which display a greenish glow. The cells are glowing because they were transfected with the cDNA from a jellyfish, Aequorea victoria, that codes for a protein called Green Fluorescent Protein (GFP). GFP is a naturally fluorescent protein; when GFP is excited with blue light at 470 nm, it emits green light at 509 nm.
The cDNA for GFP was obtained from a commercial biotech supplier (Clontech). I subcloned the jellyfish cDNA into a mammalian expression plasmid, pCEP4 (Invitrogen). This new DNA plasmid construct allows for the constituitive expression of the GFP in human cells, driven by the cytomegalovirus promoter sequence already present in the pCEP4 plasmid. The HEK 293 cells were exposed to a fine precipitate of the GFP/pCEP4 DNA construct for 24 hours, then the plasmid DNA was washed out. During this time, some cells take up the plasmids and begin to synthesize the new GFP protein. After another 24 hours, I placed the cells under a microscope equipped with epifluorescence optics for fluorescein isothiocyanate, illuminated them with blue light, and took this picture. Cells that took up the GFP/pCEP4 construct fluoresce green, while untransfected cells do not.
The green fluorescence can serve as a marker for transfection efficiency. When cells are co-transfected with a GFP-containing plasmid and another plasmid DNA, for example receptor DNA whose protein product does not glow, the success and extent of transfection can be monitored by using the green fluorescence as a reporter.
Another approach is to fuse the GFP gene to another gene of interest, say a receptor gene. The new GFP-receptor construct can then be inserted into a plasmid for transfection and expression in cells. By monitoring the green fluorescence, one can identify which cells are expressing the receptor and can determine where within the cell the receptor is being expressed. One can perform binding or functional assays to check if the receptor is working properly with the GFP protein fragment attached to it.