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

signal. The data are expressed as mean ±SD
(n=3). 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.
Figure 11. Comparison of CAT
expression from different promoters in
vitro. ELM cells (solid bars) or CFT1
cells (stippled bars), a human airway
epithelial cell line derived from a CF
patient, were transfected as described in
Figure 10. CAT ELISA assays were
carried out 48 h after transfection (an
average of 6 assays). CAT protein levels
were normalized to pCF1-CAT (in A) or
pCMVHICAT (in B). A. Expression
from plasmids containing the BGH
poly(A) signal. SPC, Surfactant protein
C promoter; NOS, nitric oxide synthase
promoter; UbB, ubiquitin B promoter;
MUC1, mucin 1 promoter; IL8,
interleukin 1 promoter; CE, CMV
enhancer; pCAT control is a
promoterless CAT plasmid. B.
Expression from plasmids containing the
SV40 poly(A) signal. CC10, Clara cell
10 kDa protein promoter; E1a,
adenovirus E1a promoter. The data are
expressed as mean ±SD (n=3-12). 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.
Figure 11 compares the strength of different
promoters from CAT constructs containing the bovine
growth hormone (BGH) poly(A) signal (panel A) or the
SV40 poly(A) signal (panel B) and the hybrid intron.
The promoters were chosen for lung targeting. CMV
yielded the highest expression in vitro. To determine
whether or not incorporating two CMV enhancers could
produce higher levels of CAT expression than one, a
Boulikas: An overview on gene therapy
32
second CMV enhancer (from -118 to -522 relative to the
transcription start site) was inserted 186 bp upstream of
the CMV promoter and its associated enhancer; in the
context of the SV40 poly(A) signal the second CMV
enhancer (CE in Figure 11) increased expression 3-fold;
however, when the BGH poly(A) signal was present, the
second copy of CMV did not increase CAT expression
(Figure 12).