Frequently Asked Questions Related to Biosafety using pGIPZ and TRC Lentiviral Vectors
NOTE: The following information pertains to lentiviral vectors (pGIPZ and TRC). If you have biosafety-related questions regarding the retroviral vector (pSM2), please contact the RNAi Core Facility via email at firstname.lastname@example.org.
pGIPZ and TRC lentiviral vectors (from Open Biosystems)What envelope glycoprotein will be expressed on virus particles?
The vesicular stomatitis virus G protein (VSV-G).Will viral particles be able to infect human cells?
Yes.What marker is used for selection/identification of infected cells?
Puromycin and GFP (pGIPz); puromycin (TRC).Is the construct replication competent?
NoIf replication defective:
What genes are deleted/modified resulting in replication defect?
A third generation lentiviral packaging system is used for generation of viral particles. This multi-plasmid approach means that no single plasmid contains all the genes necessary to produce packaged viral particles. Furthermore, the U3 portion of the 3’ LTR is deleted to eliminate the promoter-enhancer region, which further reduces the possibility of viral replication. The three plasmids comprising this system are:
1. The packaging vector, which contains the minimal set of lentiviral genes required to generate the virion structural proteins and packaging functions.
2. The vesicular stomatitis virus G protein (pCMV-VSV-G) envelope vector, which provides the heterologous envelope for pseudotyping,
3. The shRNA vector, which contains the sequence of interest as well as the cis-acting sequences necessary for RNA production and packaging. What elements of the original virus genome are retained?
Only gag, pol, rev, tat and vpr. What is the packaging system/cell line used to produce virus stocks?
293T cells.How many recombination events are necessary to produce replication competent virus (RCV)?
>3. There is no evidence that recombination events occur. Note that production of viral particles uses a well-established packaging system and established protocols, with strict BL2 level guidelines. What assay is used to test for RCV?
Serial dilution and p24 ELISA assay using cell lines that are able to propagate wild-type HIV, such as SupT1 lymphocytes.How often are virus stocks tested for RCV?
The UMMS RNAi Core Facility does not keep virus stocks, but instead prepares fresh virus from plasmid DNA. If an individual lab keeps the virus stock for an extended period of time, it will need to be tested every 6 months using the serial dilution and p24 ELISA assay described above.