Microscopic Imagery of Mammalian Cells Expressing an Enhanced Green Fluorescent Protein Gone

The green fluorescent protein (GFP; 1 –5 ) from the jellyfish Aequorea Victoria has emerged as an important reporter for monitoring gene expresston, protein localization, cell transformation, and cell lineage in VIVO and in real time. Unlike other bioluminescent reporters, the chromophore in GFP is mtrinsic to the primary structure of the protein, and GFP does not require additional factors other than molecular oxygen (see Note 2 ) to fluoresce (6 , 7 ). GFP emits bright green light (λ_max = 510 nm) when excited with ultraviolet (UV) or blue light (λ_max = 395 nm, minor peak at 470 nm). Full-length GFP (238 ammo acids; 27 kDa) appears to be required for fluorescence. However, the mimmal chromophore responsible for light absorption conststs of a Ser65-dehydroTyr66-Gly67 cyclic tripeptide, which is postulated to be buried inside the folded protein (6 ). GFP fluorescence is stable (see Note 5 ), species-independent, and can be monitored noninvasively in livmg cells by either fluorescence microscopy, flow cytometry, or macroscopic imaging technrques. GFP has been used as a reporter in a wide range of species, including a number of different mammalian cell lines (Table 1 ). Moreover, a variety of N-and C-terminal protein fusions with GFP have been constructed, and shown to maintain both the fluorescence properties of native GFP and the biological function of the fusion partner (5 , 8 –12 ).

Cell line	Cell type	Reference
293	Transformed primary embryonic kidney, human	12 , 29
BHK-21	Hamster	10
CHO-K1	Ovary, Chmese hamste	5 , 10 , 30
COS-7	Kidney, SV40 transformed, African green monkey	10 , 25
GH3	Pituitary tumor, rat	31
HeLa	Epitheloid carcinoma, cervix, human	5 , 11 , 25
JEG	Placenta, human	32
NIH/3T3	Embryo, contact-inhibited, NIH Swiss mouse	5 , 10 , 25
PtK1	Kidney, kangaroo rat	10