SPPLEMENTAL METHODS
Recombinant HSV vectors. In order to obtain the gC promoter, genomic DNA from
HSV-1 strain 17 was digested with two restriction enzymes, PstI and BamHI. Plasmid
pSKgC(17) was generated by inserting the 2.46-kb PstI-BamHI fragment that contains
UL44 (glycoprotein C, gC) into pBluescript II SK(-) plasmid (Stratagene, La Jolla, CA).
The promoter region of UL44 (gC), including the initiation element (Inr), the TATA box
and DAS
binding factor sites,
is
described in
(http://darwin.bio.uci.edu/~faculty/wagner).
E.K.
Wagner’s homepage
The gC promoter was amplified from
pSKgC(17) with the following pair of primers: forward primer (T7 primer: 5’GTAATACGACTCACTATAGGGC-3’)
and
reverse
primer
(5’-
GTCGACGCGTGCCCGACGCCTCCCCCTCGC-3’), which contains SalI and MluI
sites at the 5’ end. The amplified gC promoter fragment (0.5 kb) was electrophoresed
and extracted from the gel by using QIAquick gel extraction kit (QIAGEN, Valencia,
CA).
The IE4/5 promoter was obtained from the pHGCX HSV amplicon vector
plasmid.1 Firefly luciferase (Fluc) and Renilla luciferase (Rluc) cDNAs were obtained
from pGL3-Basic Vector and phRluc/SV40 Vector (Promega, Madison, WI), respectively.
Each luciferase gene was combined with either IE4/5 promoter or gC promoter,
generating four shuttle plasmids, pT-gC-Fluc, pT-gC-Rluc, pT-IE4/5-Fluc, and pT-IE4/5Rluc. The methods for engineering recombinant HSV (HSVQuik system) have been
previously described.2,3 Briefly, each shuttle plasmid and FLP-expressing helper plasmid
(pFTP-T) were electroporated into bacteria containing the HSV-BAC construct
(fHsvQuik-1). The resulting co-integrates of fHsvQuik-1 and the shuttle plasmids were
then co-transfected into Vero cells with the CRE-expressing plasmid (pc-nCre). This
generated the four recombinant viruses: rQ1-γ2-Fluc, rQ1-γ2-Rluc, rQ1-α-Fluc and rQ1α-Rluc. The green fluorescent protein (GFP) cDNA was inserted in-frame with the
remaining 5’-coding sequence of the deleted UL39 (ICP6) sequence, thus generating
recombinants that express GFP as a marker of infection. Viruses were propagated and
titered in Vero cells as previously described.2
REFERENCES
1
2
3
Saeki Y, Breakefield XO, Chiocca EA. Improved HSV-1 amplicon packaging
system using ICP27-deleted, oversized HSV-1 BAC DNA. Methods Mol Med
2003; 76: 51-60.
Kambara H, Okano H, Chiocca EA, Saeki Y. An oncolytic HSV-1 mutant
expressing ICP34.5 under control of a nestin promoter increases survival of
animals even when symptomatic from a brain tumor. Cancer Res 2005; 65: 28322839.
Terada K, Wakimoto H, Tyminski E, Chiocca EA, Saeki Y. Development of a
rapid method to generate multiple oncolytic HSV vectors and their in vivo
evaluation using syngeneic mouse tumor models. Gene Ther 2006.