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