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.