VSV Gene Expression Vector
Recombinant vesicular stomatitis virus (VSV) vectors are widely used for a variety of applications including studying mechanisms of viral entry into host cells, identification of cellular receptors utilized by viruses for cell entry, screening of viral entry inhibitors and vaccine development research. VSV vectors can be efficiently pseudotyped with envelop glycoproteins derived from heterologous viruses, such as envelop proteins of viruses requiring high-level containment, which can then be safely handled in a regular biosafety level 2 (BSL2) facility due to their inability to undergo more than a single round of replication. This makes VSV vectors highly suitable for studying cell entry mechanisms of such high-risk viruses without the requirement of high-level containment.
Wildtype VSV belonging to the family Rhabdoviridae has a negative-sense RNA genome whose replication relies on the viral RNA dependent RNA polymerase (RdRp). The single-stranded RNA genome encodes the following five viral proteins: nucleocapsid (N), phosphoprotein (P), matrix (M), glycoprotein (G) and the large subunit of the viral RdRp (L). The RNA genome is encapsidated by the N protein to form the ribonucleoprotein complex (RNP) inside the VSV virion. The P and L proteins constitute the viral RdRp which remains associated with the RNP. During infection, VSV enters the host cell by endocytosis which involves the fusion of the viral envelop with the endosomal membrane mediated by the G protein leading to the release of the RNP along with the associated viral RdRp into the cytoplasm. The viral RdRp immediately initiates primary transcription using the RNP as a template, and the transcribed sense RNA is translated into viral proteins required during later stages of the infection process including viral genome replication, secondary transcription, virus assembly and budding.
The recombinant VSV generated by VectorBuilder is deficient in replication due to deletion of the glycoprotein G gene. A transgene which is typically a reporter gene such as a fluorescent protein gene, a luciferase gene or a secreted alkaline phosphatase gene can be inserted at the glycoprotein G deletion site to facilitate the analysis of virus infectivity. The generation of VSV pseudotypes consists of three main steps: 1) primary recovery of VSV from plasmid; 2) generation of G-complemented VSV; and 3) pseudotyping VSV with heterologous viral envelop protein. A VSV-delta G vector is first constructed as a plasmid in E. coli which expresses the antigenomic sense RNA of VSV excluding the glycoprotein G gene and serves as the template for genomic RNA synthesis by viral RdRp during the primary recovery of recombinant VSV. To recover VSV from plasmid, the VSV-delta G plasmid is transfected along with four helper plasmids expressing the VSV N, P, G and L proteins into cells infected with recombinant vaccinia virus which expresses bacteriophage T7 RNA polymerase to transcribe the VSV-delta G plasmid DNA into antigenomic sense RNA. Recombinant VSV can be packaged and released from packaging cells. Supernatant collected from these cells is then applied to a new dish of cells transfected with a plasmid expressing VSV G protein to generate and amplify G-complemented VSV stocks. The VSV pseudotypes can then be generated by infecting cells that have been transiently transfected with a plasmid expressing the heterologous viral envelop protein with G-complemented VSV.
For further information about this vector system, please refer to the papers below.
Our VSV vector lacks the VSV envelop glycoprotein G gene and can be used to generate VSV pseudotypes with envelop proteins derived from heterologous viruses by supplying the desired envelop protein in trans. The cell tropism of recombinant VSV produced from our vector is determined by the viral envelop protein used for pseudotyping it. The VSV vector has been optimized for high copy number replication in E coli and can be customized to express any desired reporter gene to facilitate the assessment of pseudotyped VSV infectivity into target cells based on the reporter gene activity.
High viral titer: Our VSV vector can be packaged into high titer virus. The titer can reach >108 infectious units per ml (IU/ml). At this titer, transduction efficiency for cultured mammalian cells can approach 100% when an adequate amount of virus is used.
Easy to propagate: Recombinant VSV vectors can be very easily propagated in nearly all mammalian cells.
Flexibility to alter tropism: Our VSV vector lacks the VSV envelop G gene which can be replaced by any desired reporter gene. In addition, envelop glycoproteins derived from heterologous viruses can be used to pseudotype the VSV virions, which in turn ultimately determines the cell tropism of the pseudotyped VSV.
Low risk of host genome disruption: Upon transduction into host cells, VSV genome undergoes replication in the cytoplasm and does not integrate into the cellular genome. The lack of integration into the host genome can be a desirable feature for in vivo human applications, as it reduces the risk of host genome disruption that might lead to cancer.
Safety: The infectivity of recombinant VSV containing envelop protein supplied in trans is limited to a single round of replication in infected host cells. As a result, VSV vectors pseudotyped with envelop glycoproteins of high-risk group viruses (such as Ebola, Zika, Nipah, coronavirus etc.) can be safely handled in a BSL-2 containment.
Ideal as vaccine candidates: The ability of VSV to induce both humoral and cellular immune responses in vivo, the low prevalence of VSV seropositivity in the general human population and being only mildly pathogenic to humans make recombinant VSV vectors ideal for the development of human vaccines.
Limited cargo space: VSV has a genome size of 11 kb. Exceeding this size by insertion of long or multiple transgenes causes a modest reduction in viral titer which may not be desirable for most applications.
Poor infection model: Since recombinant VSV can undergo only a single round of infection in host cells, they represent a poor model for studies intended at dissecting the mechanisms underlying the actual viral infection process.
Technical complexity: The production of VSV pseudotypes involves multiple steps including recovery of VSV from plasmid, generation of G-complemented VSV and pseudotyping VSV with heterologous viral envelop proteins. These procedures are technically demanding and time consuming relative to conventional plasmid transfection.
CMV promoter: Human cytomegalovirus immediate early promoter. It can drive high-level transcription of the downstream viral antigenomic RNA in packaging cells.
T7 promoter: Promoter from T7 bacteriophage. The T7 RNA polymerase from the phage recognizes this promoter to drive high-level transcription of the downstream viral antigenomic RNA.
VSV-N: Nucleoprotein gene of vesicular stomatitis virus. Assembles into an intact RNase-resistant nucleocapsid as N/RNA polymer; required for initiating viral genome synthesis.
VSV-P: Phosphoprotein gene of vesicular stomatitis virus. Functions as a critical component of the VSV polymerase complex by positioning the large (L) protein on the N/RNA template and stimulating viral RNA synthesis with the L protein at both the initiation and elongation steps.
VSV-M: Matrix protein gene of vesicular stomatitis virus. Associates with the N/RNA nucleocapsid and facilitates virus assembly by promoting condensation of the ribonucleocapsid.
Kozak: Kozak translation initiation sequence. Facilitates translation initiation of ATG start codon downstream of the Kozak sequence.
ORF: The open reading frame of your gene of interest is placed here.
Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.
VSV-L: Large polymerase protein gene of vesicular stomatitis virus. Forms RNA-dependent RNA polymerase (RdRp) complexes with the VSV phosphoprotein and catalyzes synthesis of the N, P, M, G, L mRNAs sequentially.
HDV: Hepatitis delta virus (HDV) antigenome self-cleaving ribozyme. This ribozyme, when present in the transcript, catalyzes the self-cleavage of the transcript in cis.
T7 terminator: Transcriptional termination signal from T7 bacteriophage. Allows transcription termination of RNA transcribed by bacteriophage T7 RNA polymerase.
BGH pA: Bovine growth hormone polyadenylation signal. Transcription driven by the CMV promoter is terminated within this sequence.
pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
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