The plasmid pVSV-ΔG-N/P-MCS2-2.6 encodes the antigenomic-sense (or positive-sense) RNA of a replication-restricted recombinant vesicular stomatitis virus (rVSV) in which the glycoprotein (G) gene has been deleted and replaced with a multiple cloning site (MCS-1) containing the following restriction enzyme sites (5'-MluI-KpnI-NheI-3') which are unique in the plasmid. It also contains an additional transcription start-stop sequence between the N and P genes, which has a second multiple cloning site (MCS-2) that has sequences for the enzymes (5'-AscI-NotI-AvrII-3'). These are also unique in the plasmid.
pVSV-ΔG-N/P-MCS2-2.6 can be used to insert two heterologous genes for expression from the VSV genome. For example expression of a heterologous glycoprotein from MCS-1 and expression of a cytokine or a reporter from MCS-2. When recovered, these viruses would be useful for vaccine studies, use as reporter viruses for high-throughput antiviral drug screening, or in identification of host entry factors as described in .
This plasmid is used together with plasmids encoding the VSV nucleocapsid (N), phosphoprotein (P), glycoprotein (G), and large polymerase subunit (L) to recovery VSV-G pseudotyped ΔG-virus as described in . The antigenomic RNA of ΔG- N/P VSV is expressed from the bacteriophage T7 promoter in pBluescript, which has been further modified to contain the hepatitis delta ribozyme used to generate a precise 3' end of the VSV antigenomic RNA and a T7 terminator sequence cloned between the SacII and SacI restriction sites in pBS-SK+ [2, 3].
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.
|Antibiotic Resistance:||Ampicillin or Kanamycin (please see vial label and packing slip)|
|Amount:||10 ul aliquot of plasmid in shipping vial|
|Grow in E. coli at 37 C:||Yes|
|Cloning Site 5':||5' VSV sequence joined directly to T7 promoter|
|Cloning Site 3':||3' VSV sequence joined directly to HDV ribozyme|
|Insert Size:||12,790 bp|
|Vector Backbone and Size:||pBS-SK-ΦT, 3105 bp|
|High or low copy:||High|
|Comments:||For suggested protocol, see: Whitt, MA, J. Virol. Methods, 2010. 169(2): p. 365-74.|
VSV recovery requires BHK-21 cells and T7 supplied by vaccinia virus infection.
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