Transfection efficiency of pEF3.5b-based plasmids harboring varying antibiotic resistance genetic elements. Top: synthesis of truncated elongation factor-1α (EF1A) promoters. From the full-length EF1A promoter found in plasmid pcDEF3 , various truncations were performed to shorten the promoter without sacrificing activity. The uppermost truncation eliminates an unnecessary piece of the intron . Truncations at the 5' end of the promoter to remove fragments derived from other vectors created vectors pEF3.5, pEF3.5a and pEF3.5b; only pEF3.5b expressed proteins as well as plasmids pEF3 and pcDEF3 (data not shown). Useful restriction sites within the promoter are shown. Truncations are shown with broken lines, and the amount of promoter upstream of the Sac II site is shown. The direction of each element in pcDEF3 is shown in the topmost schematic. Bottom: vectors pEF3, pEF3.5bPGKhygro, pEF3.5bpuro, pEF3.5bneoPGK, and pEF3.5bPGKpuroPGK, all harboring and expressing the bicistronic insert EGFPEMCVChFP, were transfected using polyethyleneimine into (left) 293T cells, (middle) B16 cells, and (right) mesenchymal stem cells (MSCs), and the percentage of successful EGFP expression (black bars) as well as the average fluorescence of the EGFP-expressing cells (gray bars) were reported as a percentage of that seen in cells transfected with pEF3-EGFPEMCVChFP. In B16 cells and in MSCs, plasmid pmaxGFP was also transfected to determine whether improved transfection in these cells was obtained. The actual percentage of transfection of pEF3-EGFPEMCVChFP and pmaxGFP is shown above their respective bars.