Construction and generation of Ad vectors
AdRSV.hAAT was created by inserting a SmaI/XhoI-fragment of pBS-RSVHAATbpA (DrMark Kay, Stanford, provided by Dr. Christian Hofmann, Max Delbrück Center, Berlin)
containing the promoter and cDNA of hAAT into the KpnI(blunted)/XhoI cut pAdenoVatorpromoterless (MP Biomedicals, Heidelberg, Germany). AdCMV.p21-RSV.hAAT was
constructed by first amplifying by PCR the coding region of pBS-Cip1 (ATCC, Manassas,
VA,
USA)
introducing
BamHI
and
EcoRI
sites
with
the
primers:
5’-
AGAGGATCCGCCATGTCAGAA-3’ and 5’-GCAGAATTCCTGTGGGCGGATT-3’. The
fragment was inserted into BamHI/EcoRI cleaved pBlueskriptII to give pBS-p21. The bovine
growth hormone polyadenylation signal from pBS-RSVHAATbpA was liberated with SmaI
and XhoI and cloned into EcoRV/SalI cut pBS-p21 to yield pBS-p21bpA. Subsequently the
complete expression cassette of pBS-RSVHAATbpA as XhoI-fragment was cloned into the
XhoI site of pBS-p21bpA to give pBS-p21bpA-RSVhAATbpA. A segment containing both
expression cassettes without the second polyadenylation signal was excised with SmaI/XbaI
and inserted into KpnI(blunted)/NheI cut pAdenoVator-hCMV-Intron (MP Biomedicals,
Heidelberg, Germany).
Vector plasmids were generated by homologous recombination in E.coli BJ5183 and virus
produced in 293 cells according to the manufacturers protocol (MP Biomedicals). Virus was
purified with AdenoPacks (Sartorius Stedim, Göttingen, Germany) and infectious titres
determined with Adeno-X Rapid Titer Kit or AdEasy Viral Titer Kit (Takara Clontech, SaintGermain-en-Laye, France / Stratagene, Amsterdam, The Netherlands). Viral stocks were
assayed for contamination with replication competent adenovirus by infection of HeLa cells
with subsequent PCR analysis of culture supernatant for presence of E1 sequences (modified
after 1). No contamination was detected.
1.
Ishii-Watabe A, Uchida E, Iwata A, Nagata R, Satoh K, Fan K et al. Detection of
replication-competent adenoviruses spiked into recombinant adenovirus vector
products by infectivity PCR. Molecular Therapy 2003; 8: 1009-1116.