The replication-deficient adenovirus expressing stromelysin was
constructed as described by Graham and Prevec.
14 The cDNA
for human stromelysin was obtained by reverse transcription-polymerase
chain reaction (RT-PCR) using total RNA extracted from interleukin
lα–stimulated human trabecular meshwork cells. The primers used were
designed from available sequences (GenBank accession No. X05232) and
contained restriction sites (
BamHI and
EcoRI at
each 5′ end) to facilitate the cloning. The primers used were
5′-GGATCCGAAATGAAGAGTCTTCCAATC [forward primer, −3 to 17 nucleotides
(nt) human stromelysin cDNA] and 5′-GAATTCCTTTCAACAATTAAGCCAGCT
(reverse primer, 1437 to 1417 nt human stromelysin cDNA). Each primer
contains six extra nt in its 5′ end corresponding to
BamHI
and
EcoRI restriction sites, to facilitate the cloning.
Human stromelysin cDNA nt numbering is according to the translation
initiation site (GenBank accession No. X05232). The 1452-bp PCR product
was directionally cloned into the pΔA.CMV vector,
15 which contains the first 16 map units of the adenovirus genome, with
the region between map unit 1.3 and map unit 9.4 deleted and replaced
by the CMV promoter and a multiple cloning site. The constructed
plasmid, along with pJM17, the second plasmid consisting of a complete
adenovirus-5 genome with a pBR322 insert in the E1 region, which
exceeds packaging capacity and allows plasmid replication but not
the formation of infectious viruses,
16 was
cotransfected into the 293 cells using lipofectAMINE (Gibco BRL,
Rockville, MD) following the protocol recommended by the manufacturer.
After 10 days, a single plaque was isolated, propagated in the 293
cells, and then the resultant recombinant adenovirus
Ad.CMV-
str was purified by cesium chloride gradient
ultracentrifugation. The virus with the desired gene construction was
identified by PCR, and the absence of E1 containing wild-type
adenovirus contamination was also confirmed by PCR, using primers
specific for the adenovirus E1 region as described by Sullivan et
al.
17 Viral titer was 4 × 10
10 plaque-forming unit (pfu)/ml, as determined by the 50% tissue culture
infection dose (TCID
50) method.