Supplementary Materials and Methods
Construction of Adenoviral Vector
A rat IFN-α cDNA fragment (662 bp) was amplified from rat liver RNA by RT-PCR.
Recombinant adenoviruses expressing rat IFN-α (Ad/IFN-α) or LacZ (Ad/LacZ) were
constructed using the AdEasy system1 as previously described.2 In brief, both IFN-α
and LacZ genes were inserted downstream of the cytomegalovirus (CMV) immediate
early gene promoter in a shuttle plasmid, pAdTrack/CMV. The shuttle plasmids
containing the inserts were transformed with pAdEasy-1, the adenoviral backbone
plasmid, into a recombination permissive Escherichia coli strain (BJ5183), yielding
plasmids pAd/IFN-α and pAd/LacZ, respectively. The structures of the resulting
recombinant vectors were confirmed by restriction enzyme digestion and PCR
analysis. The recombinant DNA was transfected into HEK 293 cells by calcium
phosphate precipitation methods to obtain recombinant adenoviruses. These were
further amplified and purified by BD Adeno-XTM virus purification kits (BD
Biosciences) following the manufacturer’s instruction. Virus titers were determined
by measuring the 50% tissue culture infectious dose (TICD50) ).3 The biological
activity of rat IFN-α expressed from the adenoviral vectors was verified by a
replication inhibition assay on an IFN-sensitive RNA virus, vesicular stomatitis virus
(VSV), using the conditioned medium obtained from adenovirus-infected Huh7 cells
(Figure S5).
Reverse Transcription-Polymerase Chain Reaction (RT-PCR ) and Real-time
qRT-PCR
Expression of AFP in liver tumors was analyzed by RT-PCR, whereas the expression
of TGF-α, TGF-β, and TIMP-1 in the liver and of liver-expressed IFN-α during DEN
administration was analyzed by real-time qRT-PCR using the SYBR Green method
(Toyobo, Osaka, Japan). Tissue RNA was extracted by TRIZOL (Invitrogen), and 2
μg of RNA were subjected to reverse transcription. One-tenth volume of the cDNA
product was then incubated with specific primers (Table S1). For AFP RT-PCR, two
pairs of primers were used: one pair amplified the 5’ coding region, and the other pair
amplified the 3’ coding region. The reactions (94˚C, 30 sec; 61˚C, 30 sec; 72˚C, 1 min)
were conducted for 25 cycles. For real-time qRT-PCR, the reaction mixture was
denatured at 95˚C for 2 min, followed by 40 cycles of PCR reactions with the
following settings: 95˚C, 15 sec; 60˚C, 15 sec; and 72˚C, 45 sec. The expression level
of each target gene was normalized to β-actin expression.
References
1 He, TC, Zhou, S, da Costa, LT, Yu, J, Kinzler, KW and Vogelstein, B (1998). A
simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci.
U. S. A. 95: 2509-2514.
2. Tai, KF, Chen, PJ, Chen, DS and Hwang, LH (2003). Concurrent delivery of
GM-CSF and endostatin genes by a single adenoviral vector provides a synergistic
effect on the treatment of orthotopic liver tumors. J Gene Med 5: 386-398.
3. Kanegae, Y, Makimura, M and Saito, I (1994). A simple and efficient method for
purification of infectious recombinant adenovirus. Jpn. J. Med. Sci. Biol. 47:
157-166.