Two-reporter FACS analysis of mammalian cells using green fluorescent proteins

摘要

Two spectrally distinguishable GFPs are used as reporters in mammalian cells to simultaneously and independently analyze the expressions of two transcriptional elements. The two GFPs, encoded by single stably integrated transcriptional elements, are readily and quantitatively detectable by FACS or flow cytometry. One of the GFP mutants (S202F, T203I, V163A) retains only the major excitation peak of wild-type GFP, while the other (S65T, V163A) retains only the minor excitation peak of wild-type GFP. Both variants have emission peaks overlapping that of wtGFP. The first mutant is excited at 406 nm using a Kr ion laser, while the second mutant is excited at 488 nm using an Ar ion laser. Emissions from both GFPs are measured at about 515 nm. The mutant excited at 406 nm can be used in conjuction with a fluorescein- based assay such as FACS- Gal. Applications include drug screening, measurements of temporal orders of gene expression, analysis of signal transduction pathways, and measurements of protein-protein interactions using two-hybrid systems.