Use of green fluorescent protein variants to monitor gene transfer and expression in mammalian cells.
Two mutants of the green fluorescent protein (GFP), RSGFP4 and GFPS65T, have been recently created which differ from the wildtype GFP of A. victoria in their excitation maxima. Here we show that human fibroblasts transfected with either of the two mutant GFP genes emit a green fluorescence that is 18-fold brighter than the cells transfected with the wildtype GFP gene. Retroviral vectors expressing the improved GFP gene were also constructed to determine their suitability for stable gene transduction into mammalian cells. The inclusion of the RSGFP4 gene in a retroviral vector did not reduce the viral titer and resulted in a fluorescent signal in viable transduced cells detectable by both fluorescence microscopy and fluorescence-activated cell sorter (FACS) analysis. Therefore, the improved mutant GFP provides a vital marker for monitoring gene transfer and expression in mammalian cells.
Cheng
L
L
Research Division, SyStemix, Inc., Palo Alto, CA 94304, USA. lcheng@stem.com
Fu
J
J
Tsukamoto
A
A
Hawley
R G
RG
D000818:Animals; D001709:Biotechnology; D002460:Cell Line; D005453:Fluorescence; D015870:Gene Expression; D018014:Gene Transfer Techniques; D014644:Genetic Variation; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D051379:Mice; D009154:Mutation; D012190:Retroviridae / Q000235:genetics; D014161:Transduction, Genetic; D014162:Transfection
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins