TwinGFP, a marker for cell cycle analysis in transiently transfected cells.

Green fluorescent protein (GFP) is widely used as a marker to identify transfected cells either by fluorescence microscopy or flow cytometry. However, cell cycle analysis with propidium iodide typically employs ethanol for cell permeabilization. During this treatment, soluble GFPs generally leak out of cells, probably due to their small size. We have now significantly improved cellular retention by creating an in-frame fusion of two GFP DNA sequences, thereby generating a double-sized GFP (TwinGFP, 57 kDa). Permeabilized HeLa cells transfected with pTwinGFP showed a strong green fluorescent signal localized throughout the cells that could easily be detected by fluorescence microscopy and flow cytometry, in contrast to cells transfected with a standard single GFP construct. The experiment indicates that protein size constitutes the major determinant of the loss of fluorescence in permeabilized cells. As a proof of principle, pTwinGFP was cotransfected with the p53 tumor suppressor gene into HeLa cells, and cells transiently expressing p53 could be identified and phenotypically characterized by flow cytometry.