An enhanced green fluorescent protein allows sensitive detection of gene transfer in mammalian cells.
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has become an important marker of gene expression. However, the sensitivity of wild-type GFP has been below that of standard reporter proteins, such as beta-galactosidase, which utilize enzymatic amplification. To improve the detection of GFP in transfected mammalian cells, we have constructed a unique GFP variant which contains chromophore mutations that make the protein 35 times brighter than wild-type GFP, and is codon-optimized for higher expression in mammalian cells. These changes in the GFP coding sequence provide an enhanced GFP (EGFP) that greatly increases the sensitivity of the reporter protein. We show that the EGFP expression vector delivered into mammalian cells gives rise to bright fluorescence that is readily detectable following a 16-24 hr transfection interval. Visual detection of transfected cells with EGFP appears to be more sensitive than equivalent measurements with beta-galactosidase catalyzed conversion of the X-gal substrate. We conclude that EGFP allows sensitive and convenient detection of gene transfer in mammalian cells.
Zhang
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G
Clontech Laboratories, Inc., Palo Alto, California 94303, USA.
Gurtu
V
V
Kain
S R
SR
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA; D018014:Gene Transfer Techniques; D005819:Genetic Markers; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D008969:Molecular Sequence Data
D005819:Genetic Markers; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004247:DNA