01. Gene therapy Boulikas.pdf - Gene therapy & Molecular Biology

B. Transcription factor binding sites
within the CMV promoter
Because of its wide use and the more potent effect,
the CMV IE enhancer/promoter deserves some special
attention. In order to understand the potent effect of the
CMV promoter in the expression of foreign genes we
need to understand the transcription factors (TFs) that
activate this regulatory region; TFs in the transfected cell
will be responsible for binding to the CMV promoter
leading to the activation of the transgene. At present not
all TF regulatory circuits leading to activation of CMV
have been deciphered. Figure 13 shows two CMV
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promoters retrieved from Genbank which are being used
in expression vectors.
The CMV IE promoter includes the 10-bp
palindromic sequence CCATATATGG (Figure 13)
which resembles the core motif of serum response
elements and proved to bind specifically to the cellular
nuclear protein serum response factor (SRF). Reporter
gene constructs containing four tandem copies of these
elements displayed up to 13-fold increased basal
enhancer activity and 18-fold tetradecanoyl phorbol
acetate responsiveness in U937 cells (Chang et al, 1993).
Figure 12. Effect of a second CMV enhancer region on CAT expression from the CMV promoter. Plasmids were transfected into ELM
cells and the cells were harvested 48 h after transfection. Expression was normalized to pCMVHICAT (in B). A. CAT protein levels in
cell lysates. RE, RSV LTR enhancer. B. Levels of CAT RNA. Total RNA was isolated from the transfected cells and a quantitative
RNA protection assay was performed. The data are expressed as mean ±SD (n=3-9). From Yew NS, Wysokenski DM, Wang KX,
Ziegler RJ, Marshall J, McNeilly D, Cherry M, Osburn W, Cheng SH (1997) Optimization of plasmid vectors for high-level expression
in lung epithelial cells. Hum Gene Ther 8, 575-584. Reproduced with kind permission of the authors (Nelson Yew, Genzyme Corp.,
Framingham, MA) and Mary Ann Liebert, Inc.
Two multicopy basal enhancer motifs within the
simian CMV IE enhancer, namely, 11 copies of the 16-bp
cyclic AMP response element (CRE) and 3 copies of
novel 17-bp serum response factor (SRF) binding sites