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

protein (e.g. Smith et al, 1995; Fong et al, 1995; Shalaby
et al, 1995).
Gene transfer to the embryo has shown the importance
of the promoter, large genomic regulatory regions, cellcell
interactions and gene switch taking place during
embryogenesis in maintaining transgene expression in
different tissues; results obtained in embryos reflect the in
vivo patterns of tissue-specific expression which could be
useful to direct efforts in promoter choices for somatic
gene transfer to the adult (as is the case for most gene
therapy applications). Furthermore these studies provide
the foundation of a new era where genetic manipulation of
the embryo could permanently correct monogenic genetic
disorders such as hemophilias, thalassemias and others.
The promoter of the tie gene, which encodes a receptor
tyrosine kinase that is expressed in the endothelium of
blood vessels, was used to drive the expression of a
luciferase reporter gene construct; in cultured cells the
luciferase activity was not restricted to endothelial cells. In
contrast, in transgenic mice expression of the reporter βgalactosidase
was restricted to endothelial cells
undergoing vasculogenesis and angiogenesis; in adult
transgenic mice, tie promoter activity in lung and many
vessels of the kidney was as high as in the vessels of the
corresponding embryonic tissues, whereas in the heart,
brain and liver, tie promoter activity was downregulated
and restricted to coronaries, cusps, capillaries, and arteries
(Korhonen et al, 1995).
Gene Therapy and Molecular Biology Vol 1, page 85
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A retroviral VEGF expression vector was used to
infect quail ebryo and to increase the level of VEGF
during critical periods of avian limb bud growth and
morphogenesis. Overexpression of VEGF in the limb bud
exclusively resulted in hypervascularization as reflected
by an increase in vascular density from an augmentation
of the VEGF signaling mechanism in a permissive
environment; vascular permeability was also dramatically
increased leading to local edema (Flamme et al, 1995).
An avian leukosis virus (ALV)-based retroviral vector
system was used for the efficient delivery of genes into
preimplantation mouse embryos; a subset of the integrated
proviruses expressed the delivered chloramphenicol
acetyltransferase (CAT) reporter gene either from the
constitutive viral promoter contained in the long terminal
repeat or from the internal nonviral tissue-specific
promoter in different sets of experiments. Thus, many of
the sites that are accessible to viral DNA insertion in
preimplantation embryos were thought to be incompatible
with expression in older animals (Federspiel et al, 1996).
Baldwin and coworkers (1997) have found that the
expression of lacZ gene under control of CMV or RSV
promoter transferred to early, postgastrulation mouse
embryos gave tissue-specific patterns of expression which
depended on the type of promoter used. Embryos were
injected into the mesoderm of the neural fold (A in Figure
30) and β-galactosidase activity was detected in the head