Figure 6. Compound promoter constructs can
modulate transgene expression. Vimentin (
VIM) and
CD44
evolutionary conserved regions (ECRs) were tested were tested in
various combinations. For both genes, we examined the effect of adding
gene-specific ECRs to either of the
VIMR2 or
CD44R2
element-containing vectors; in each case the added ECR was positioned
5′ to the existing cloned promoter element (
VIMR2 or
CD44R2)
within the multiple cloning site (MCS), to the, to the existing
promoter element (
VIMR2 or
CD44R2). Adding VIMR2–6 to
the VIMR2-containing construct resulted in slightly lower number of
cells expressing eGFP (shown in red), while the mean fluorescence
intensity (MFI) remained constant. When CD44R3–6 were individually
added to CD44R2 (shown in blue), both cell counts and MFIs showed a
modest decrease. The VIMR2-Reverse reduced expression to near baseline
levels. A chimeric promoter construct containing
VIMR2 (3′) and
CD44R2 (5′) elements (red/blue) did not change expression
significantly. Bars indicate fold change in number of eGFP positive
cells, normalized to the “first-generation” promoter-less parent vector
(pFTMGW) [
22].
The MFI for each construct was also normalized to the parent vector
(pFTMGW) and is shown immediately below each bar (shown in orange).
Refer to
Appendix
2 for genomic coordinates of each construct. Compared to pFTMGW,
all constructs (except
VIMR2R) showed quantitatively different
expression. p<0.01, using a two-tailed Student's
t-test
assuming equal variances. The asterisk equals p<0.05, the double
asterisk equals p<0.01, and the triple asterisk equals p<0.001,
using a two-tailed Student's
t-test assuming equal variances.
Error bars represent 1 standard deviation.