Lentiviral Delivery of Proteins Using 2A Peptides Figure 1. Evaluation of bicistronic transgene expression in transiently transfected HEK 293FT cell cultures. (A–C) Diagrams of the three bicistronic reporter transgenes analyzed in this study. The solid lines below each transgene indicate predicted mRNA transcripts. Abbreviations: W - WPRE; H - hemagglutinin tag; EF1 – elongation factor 1 promoter; GFP – green fluorescent protein; mCher – cherry fluorescent protein; 2A - porcine teschovirus (pTV1) 2A-like cleavage peptide. (D–G), (H–K) and (L–O) are representative images taken of HEK 293FT cell cultures that were transiently transfected with the lentivector plasmids carrying the indicated transgenes 48 h post-transfection. Image exposure times: GFP images in panels D,H and L = 17 ms; mCherH images in panels E, I and M = 4 ms; Hoechst images in panels G, K and O = 5 ms. Scale bar in panel D = 100 mm. (P–S) Western blot analyses of the expression of the GFP and mCherH reporter proteins encoded by the bicistronic transgenes. Three replicate HEK 293FT cultures were analyzed for each transgene and one culture was analyzed for each of the control vectors, EF1-mCherH (CH) and EF1-GFP (GFP). Four identical western blots containing the protein samples were generated and each was probed with an antibody recognizing either hemagglutinin (P), GFP (Q), 2A (R), or GAPDH (S). GAPDH was used as a protein loading control. (T) Densitometric analyses of GFP and mCherH protein levels produced from each transgene Significant differences (*p,.05) between the normalized mean GFP and mCherH values are indicated with brackets. (U) The cleavage efficiency of GFP-2A-mCherH was estimated by expressing the intensities of the cleaved GFP-2A (Q) or mCherH (P) protein bands as a percentage of total GFP or mCherH immunoreactivity (intensity of the cleaved plus uncleaved protein bands), respectively. Each estimate is the mean6SD of three samples. doi:10.1371/journal.pone.0020553.g001 transfected HEK 293FT cells by monitoring expression of GFP and mCherH using both fluorescent microscopy and western blot. Fluorescent imaging of live cells was used to determine if GFP and mCherH could be detected in transfected cells and if the relative amounts of these two proteins were uniform among transfected cells. Comparisons of GFP (Figure 1D,H,L) and mCherH (Figure 1E,I,M) expression in transfected cultures indicated that the levels of GFP in cultured cells expressing the EF1-GFP-EF1-mCherH transgene were significantly lower than those expressing the other two transgenes while expression of mCherH from all three transgenes appeared to be similar. Examination of the merged GFP and mCherH images (Figure 1F,J,N) revealed that expression of GFP and mCherH was most uniform across cells in cultures expressing the EF1- GFP-2A-mCherH transgene; nearly all of the transfected cells appeared yellow (Figure 1N). In cultures expressing the EF1- GFP-W-EF1-mCherH transgene, expression of GFP and mCherH across cells was moderately uniform; transfected cells were either green or yellow (Figure 1J). Expression of GFP and mCherH was least uniform across cells in cultures expressing the EF1-GFP-EF1-mCherH; in these cultures, transfected cells ranged from green to red (Figure 1F). PLoS ONE | www.plosone.org 3 May 2011 | Volume 6 | Issue 5 | e20553