Promoters and Plasmids
While cationic polymers and lipids play an important role in delivering genes to cells, promoters play an equally important role in ensuring the expression of the gene that must not be overlooked. Indeed, transgene expression has been shown to vary significantly for different promoters between cell types.33,34 For example, the mammalian EF1α promoter provides high levels of transgene expression in mouse embryonic cell lines, while expression from the viral cytomegalovirus (CMV) promoter is significantly lower in those cells.35
Figure 2 shows a comparison of three commonly used promoters (EF1α, CMV, and the CMV early enhancer/chicken beta-actin/rabbit beta globin hybrid promoter known as “CAG”) that were used to drive expression of luciferase (Figure 2A) and EGFP (Figure S1) in Jurkat T cells. Similar trends in luciferase expression were observed with both Lipofectamine and jetPEI, with the EF1α promoter showing the highest luciferase levels followed by a slightly lower (but not statistically significant) decrease in the level of luciferase expression provided by the CAG promoter. A significantly lower level of luciferase expression was observed with the viral CMV promoter, which was the only promoter in these experiments that lacked an intron. This may explain the decrease in luciferase expression levels, since introns have been previously shown to increase transgene expression in other cell types.36 However, it is also interesting to note that no significant difference in GFP expression was observed between the promoters (Figure S1).
Effects of Cell Culture Media
In addition to vehicles and promoters, the effects of several other variables were also tested to optimize transfection efficiency in Jurkat T cells. For example, cells were treated with small molecule inhibitors that have previously been shown to enhance transgene expression in other cell types (e.g., BX795, MS-275, AG-490, HMN-214 et al).37,38 One of the inhibitors (iCRT14) enhanced luciferase expression approximately 3-fold, but did not significantly enhance transfection efficiency (i.e., %EGFP+ cells, shown in Figure S2). We also compared the conventional pEF-GFP plasmid to EGFP-expressing nanoplasmids (Nature Technologies, #NTC9385R), which are much smaller (1,594 bp) than pEF-EGFP (5,051 bp) and have been shown to increase transfection efficiency and the duration of EGFP expression in other cell lines and in vivo .39,40 However, at both 24 and 48 hour time points following transfection, no significant differences in EGFP expression were observed between pEF-GFP and the nanoplasmids in Jurkats. Finally, varying amounts of Lipofectamine did not have a significant effect on transfection efficiency was observed (Figure 3A), but the lowest dose of Lipofectamine was significantly less toxic to the Jurkat T cells (Figure 3B).
One variable that did significantly influence transfection efficiency was the type of media used during the transfection. For example, the initial experiments shown in Figures 1 and 2 were conducted in serum-containing RPMI-1640 media, since that is the media recommended by ATCC for Jurkat cells (clone E6-1, TIB-152). In contrast, chemically-defined serum-free media formulations like X-VIVO™ 15 are typically used in primary T cell culture to avoid the potentially problematic effects of serum components in vivo . As shown in Figure 3C, culturing Jurkat cells in X-VIVO media significantly increased the transfection efficiency of Lipofectamine in Jurkat cells (63.0±10.9% EGFP+), relative to the modest transfection efficiency observed in RPMI media (23.1±5.5% EGFP+). Similar results were also observed with transfections of nanoplasmids (Figure S3).
Since albumin and other components present in serum-containing (SCM) RPMI are known to inhibit transgene delivery,41,42additional transfections were performed with serum-free (SFM) RPMI and serum-containing X-VIVO media. The exclusion of serum from RPMI had no significant effect on transfection efficiency, but the addition of serum to the X-VIVO media did significantly decrease transfection efficiency. However, the transfection efficiency obtained with serum-containing X-VIVO media was still higher than both SCM- and SFM-RPMI at 24 and 48 hours post-transfection, which suggests that some component of the X-VIVO media may enhance transfection. For example, one component that is present in X-VIVO but absent in RPMI is recombinant transferrin, which has previously been shown to increase Lipofection efficiency by enhancing endocytosis and nuclear targeting.43,44