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General Questions
What is the safety concerns associated with using retroviral vectors and packaging cells?
    The National Institute of Health and the Center for Disease Control have designated retroviral vectors such as those from Moloney murine leukemia virus (MoMuLV) as Biosafety Level 2. NIH guidelines require that retroviral production and infection be performed in a Biosafety Level 2 facility.
    Recombinant retroviruses are extremely labile and are inactivated by ethanol, detergents, or bleach Because of their lipid-derived membrane.
Why use a retroviral expression system?
    Expression systems based on retroviral gene delivery are generally more reliable and have broader utility than standard plasmid-based transfection systems. Retroviral gene delivery (infection) is usually used instead of transfection when cells are difficult to transfect. Retroviruses preferentially integrate into actively transcribed regions of the genome. The infection yields a cell line that stably expresses your gene of interest.
What is the limit on the size of my DNA insert?
    Retroviruses efficiently package RNA that is approximately 8-9 kb. Most developed vectors can take as large as ~6 kb and still be packaged with no reduction in viral titer.
Is the poly A + sequence needed when cloning my gene of interest into a retroviral vector?
    You do not need to include the poly A + sequence with your gene because the native polyadenylation signal (from the wild-type virus) that is located in the 3' LTR of most retroviral vectors is sufficient for polyadenylation of the transcribed gene of interest.
If I transfect or infect with an EGFP construct, when will I begin to observe fluorescence?
    Enhanced green fluorescent protein (EGFP), often used to check transfection efficiency or viral titer, can be visualized in packaging cells 24-48 hours after transfection. In target cells, EGFP is usually detected 48-96 hours after infection, depending on the cell type and infection efficiency.
Can I freeze the viral supernatant and use it later?
    Yes. Collect the virus at 48 hours after transfection, filter with a 0.45µm cellulose acetate or polysulfonic filter, and then freeze at –80°C. You do not need to add a cryopreservative such as glycerol or DMSO. When you are ready to use the frozen supernatant, thaw it quickly by placing it in a 37°C water bath until ice crystals have just disappeared.