Biomedical Engineering Reference
In-Depth Information
3
Methods
3.1 Vector
Construction
and Production
HSV-1 has a number of biological features that make it attractive
as a gene delivery vehicle to the nervous system [
1
,
8
,
9
]. One
important feature is its large genome (152 Kb) allowing many viral
genes to be removed and replaced by large or multiple transgenes
[
10
,
11
]. HSV genes are expressed in a sequential order during a
lytic replication cycle, with immediate early genes (IE) initiating
the cascade of coordinated viral gene expression. The replication-
defi cient vectors used in our studies were generated from a vector
backbone deleted for the essential IE gene ICP4 which blocks the
expression of later genes in the gene expression cascade [
12
,
13
].
We fi rst engineered the DPZ vector (Fig.
2a
) in order to express
the
Escherichia coli lac
Z gene driven by the ubiquitous
human cyto-
megalovirus
(HCMV) promoter to study the transduction pattern
of this vector in the brain after its injection into the DRt. Then we
constructed another set of vectors (THz, THa, THTH) which
already carry the HCMV promoter and the enhanced green fl uo-
rescent protein (EGFP) cDNA, inserted between UL36 and UL37
Fig. 2
Schematic representation of the HSV-1 constructs DPZ and THa. The vectors do not express the viral
genes ICP4 (
ICP4; deletion) and thymidine kinase (TK, UL23; insertional inactivation). (
a
) The DPZ vector car-
ries a cassette, inserted into the TK gene, for the expression of the
E. coli lacZ
gene driven by the HCMV
immediate early enhancer-promoter. (
b
) The THa vector carries a cassette inserted into the TK gene, contain-
ing the TH cDNA inserted in antisense orientation relative to the rat TH promoter, and a second cassette,
inserted between the UL36 and UL37 viral genes, for the expression of the enhanced green fl uorescent protein
(EGFP).
PA
polyadenylation signal,
TR
terminal repeat,
IR
internal repeat,
L
long segment,
S
short segment
∆
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