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Green Fluorescent
Protein (GFP)
Green fluorescent protein (GFP) has recently become a valuable and favoured tool for the rapid detection of gene transfer. This is because the marker can be visualised without interruption or termination of an experiment as is required with the detection of other commonly used markers such as ß-galactosidase. GFP is a 238 amino acid polypeptide that acts as an energy-transfer acceptor under physiological conditions in the jellyfish Aequorea victoria deriving excitation energy from emission of blue light via a calcium binding-activated photoprotein, aequorin. GFP is capable of producing a strong green fluorescence when excited by blue light (without the need for a substrate), which can be monitored using standard ultraviolet microscope technology. GFP does not appear to interfere with cell growth and function. Expression is stable for at least 10min and persists after fixation with formaldehyde, but is hindered by ethanol {7, 17} GFP expression allows the isolation of stable cell lines expressing an uncharacterized protein for which there is no specific antibody or in which an epitope tag would produce an altered function. The protein has been targeted to specific organelles using a fused targeting leader sequence {8,12}. Mutation of Ser65 (part of the ºp-hydroxybenzylideneimidazolinone chromophore) to Threonine (S65T) produced an increase in excitation amplitude of four to six times the wild-type, four times more quickly than the wild-type, as well as large shifts in the excitation maxima (from peaks of 396 and 475nm to 470-490nm) that make GFP visible in fluorescence activated cell sorting (FACS) {10}. Detection in log phase bacteria at 37°C was possible within eight minutes compared with one to three hours using wtGFP {4} with a red-shifted maximum. The S65T gfp was then "humanised" (hgfp) by introducing 92 silent mutations that alter the codons to those more commonly used in human genes. This hgfp has been placed under the control of a CMV immediate-early promoter in the retroviral backbone pLNCX and transduction traced successfully {20}. It has recently been reported that a significantly higher frequency of rearrangement of proviral sequences occurs in stable clones compared to control producer cells. G418 stable clones producing GFP could not be isolated after drug selection despite initial detection of GFP in the packaging cell lines (which died within days to weeks). Selective pressures lead to an advantage through the abrogation of GFP expression, but the retention of neo which was necessary for growth under selection {405}. References |
