Abstract:
:Green fluorescent protein (GFP) is widely used as a reporter gene in both prokaryotes and eukaryotes. However, the fluorescence levels of wild-type GFP (wtGFP) are not bright enough for fluorescence-activated cell sorting or flow cytometry. Several GFP variants were generated that are brighter or have altered excitation spectra when expressed in prokaryotic cells. We engineered two GFP genes with different combinations of these mutations, GFP(S65T,V163A) termed GFP-Bex1, and GFP(S202F,T203I,V163A) termed GFP-Vex1. Both show enhanced brightness and improved signal-to-noise ratios when expressed in mammalian cells and appropriately excited, compared with wtGFP. Each mutant retains only one of the two excitation peaks of the wild-type protein. GFP-Bex1 excites at 488 nm (blue) and GFP-Vex1 excites at 406 nm (violet), both of which are available laser lines. Excitation at these wavelengths allows for the independent analyses of these mutants by fluorescence-activated cell sorting, permitting simultaneous, quantitative detection of expression from two different genes within single mammalian cells.
journal_name
Proc Natl Acad Sci U S Aauthors
Anderson MT,Tjioe IM,Lorincz MC,Parks DR,Herzenberg LA,Nolan GP,Herzenberg LAdoi
10.1073/pnas.93.16.8508subject
Has Abstractpub_date
1996-08-06 00:00:00pages
8508-11issue
16eissn
0027-8424issn
1091-6490journal_volume
93pub_type
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