Vaccinia Virus rVV-18R-T7
rVV-18R-T7 is a recombinant vaccinia virus (rVVs) that encodes the bacteriophage T7 RNA polymerase and has the B18R (soluble type I interferon receptor) gene deleted [1, 2]. These rVVs are used for transient, high-level expression of cDNAs under the transcriptional control of T7 promoters, such as in the plasmid pBS. The viruses are also used for the recovery of recombinant vesicular stomatitis virus (rVSV) using reverse genetics [3-6].
It is the responsibility of the principal investigator to seek Institutional Biosafety Safety Committee approval for recombinant DNA, transgenic animal or infectious agent use within their laboratory spaces and maintain an Institutional Biosafety Safety Committee approval during the time period these materials are used.
From the laboratory of Michael A. Whitt, Ph.D., University of Tennessee.
Part of The Investigator's Annexe program.
This product is only for sale in the US. KeraFAST requires a copy of your USDA APHIS VS 16-6 or 16-6A permit prior to shipment be sent here.
||rVV-18R-T7; Replication-competent, attenuated vaccinia virus
||Western Reserve (WR)
||The gene for T7 RNA polymerase was subcloned into the plasmid pSC11 under control of the vaccinia p7.5 early/late promoter to generate pSC-T7. This plasmid also contains the E. coli LacZ gene under control of the vaccinia p11 late promoter . Both T7 polymerase and LacZ are flanked by sequences from the vaccinia thymidine kinase gene. Recombination of pSC-T7 into either VV-B18R or VV-B15R resulted in rVV-T7 viruses that were initially plaque-purified using positive blue-white screening, and then T7 RNA polymerase expression was confirmed by Western blot analysis.
||~1 x 109 plaque forming units/ml
||To produce working stocks, infect HeLa or BSC-40 cells at a multiplicity of 0.1 to 0.5 and incubate for 2-3 days, or until 95% of the culture showed CPE. Harvest and purify as described in .
To recover recombinant VSV by reverse genetics, infect BHK-21 cells at a multiplicity of 3 to 5 and transfect with plasmids encoding a VSV genome (e.g. pVSV-FL+-2 or pVSV-ΔG) and support plasmids expressing the VSV N, P, and L proteins (and VSV G protein if recovering a ΔG virus as described in .
||Store at -80C. Multiple freeze/thaw cycles not recommended.
||Frozen on dry ice
||It is the responsibility of the principal investigator to seek IBC approval for recombinant DNA, transgenic animal or infectious agent use within their laboratory spaces and maintain an IBC approval during the time period these materials are used.
USDA APHIS VS 16-6 or 16-6A permit must be obtained and a copy of the permit must be sent to KeraFAST in advance of shipment. The Application Form VS 16-3 (Import controlled material import or transport organisms or vectors) must be submitted to USDA APHIS Veterinary Services to obtain the VS 16-6 or 16-6A permit.
These are replication-competent viruses that grow to high titers in HeLa and BSC-40 cells (as well as most other mammalian cell types). T7 RNA polymerase expression is easily detectable by Western blot 1 hr post-infection, and transient expression of genes under control of a T7 promoter on plasmids transfected into rVV-T7 infected cells is detectable by immunofluorescence staining as early as 3 hours post-transfection.
- Alcami, A. and G.L. Smith, A soluble receptor for interleukin-1 beta encoded by vaccinia virus: a novel mechanism of virus modulation of the host response to infection. Cell, 1992. 71(1): p. 153-67.
- Symons, J.A., A. Alcami, and G.L. Smith, Vaccinia virus encodes a soluble type I interferon receptor of novel structure and broad species specificity. Cell, 1995. 81(4): p. 551-560.
- Jayakar, H.R., K.G. Murti, and M.A. Whitt, Mutations in the PPPY motif of vesicular stomatitis virus matrix protein reduce virus budding by inhibiting a late step in virion release. J. Virol, 2000. 74(21): p. 9818-9827.
- Lawson, N.D., et al., Recombinant vesicular stomatitis viruses from DNA. Proc.Natl.Acad.Sci.(USA), 1995. 92(10): p. 4477-4481.
- Whelan, S.P.J., et al., Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones. Proc. Natl. Acad. Sci. USA, 1995. 92: p. 8388-8392.
- Whitt, M.A., Generation of VSV pseudotypes using recombinant DeltaG-VSV for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines. J. Virol. Methods, 2010. 169(2): p. 365-74.
- Chakrabarti, S., K. Brechling, and B. Moss, Vaccinia virus expression vector: Coexpression of β-galactosidase provides visual screening of recombinant virus plaques. Mol.Cell.Biol., 1985. 5: p. 3403-3409.
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