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viral promoter since the 5' LTR is ultimately replaced by the deleted 3' LTR
during viral replication. Thus, gene expression is instead driven by an internal
promoter. Absence of enhancer and promoter sequences in both LTRs of the
integrated provirus minimizes the possibility of activating cellular proto-
oncogenes and provides a safer vector for gene therapy
(6)
. Prolonged expres-
sion of SIN vector was attributed to the lack of methylation of the LTR and to
the absence of a heterochromatin-induced inactivation of transcription, which
occurs frequently in the integrated viral sequence in mammalian cells
(7
,
8)
.
An important feature of the gene delivery system is the ability to regulate
the expression of a delivered gene. Efficient delivery of regulatable genes by
retroviral vectors makes it possible to analyze the population of transduced
cells, circumventing a lengthy process and clonal variation, often observed in trans-
fection of plasmids in mammalian cells. Several inducible gene-expression
systems such as those controlled by heat shock, steroids, or metallothionein
suffer from either high basal levels of gene expression or pleiotropic effects
on host cell genes
(9)
. Tetracycline (Tc) -inducible gene expression is restricted
to the regulation of the gene of interest only because DNA response elements
were derived from
Escherichia coli
, thus preventing pleiotropic effects on host
cell genes.
The tetracycline-resistance operon of the Tn10 transposon are negatively
regulated by the Tet repressor (
TetR
), which blocks transcription by binding to
the tet operator sequences (
tetO
) in the absence of Tc
(10
,
11)
. There are two
components in the Tc-inducible system. The first one is a hybrid regulatory
protein base on
TetR
under the control of the
SV40
promoter. To convert
TetR
from a repressor into a Tc-controlled transactivator, amino acids 1-207 were
fused to the C-terminal 127 amino acids of the VP16 protein of herpes simplex
virus
(10
,
11)
. The hybrid protein binds to the tetracycline-responsive element
(TRE) and thereby activates transcription in the absense of Tc (Tet-off system).
The second component is the response element which expresses the gene of
interest, cloned in the multiple cloning site, under the control of TRE. The
TRE consists of seven copies of the 42-bp
tetO
sequence and is located
upstream of the minimal immediate early promoter of CMV. These two com-
ponents can reside in the same retroviral vector or can be divided into two
retroviral vectors
(11
,
12)
. Regulation of gene expression can be turned off by
Tc (Tet-off system) as described above or turned on by Tc (Tet-on system).
The four amino acid changes in
TetR
results in a “reverse” Tet repressor
(
rTetR
), which binds the TRE sequence in the presence of Tc
(11
,
13)
. There-
fore, when fused to the VP16 activation domain,
rTetR
activates transcription
in the presence of Tc (Tet-on).
Selection of the retroviral vectors will depend on the system where gene
expression will be studied. In general, constitutive vectors are likely to yield a
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