Transfected cells
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.
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