Q: What is the required biosafety level for using recombinant viruses?
Q: Will adenovirus/lentivirus/retrovirus work well in my cell models?
Q: How should I prepare DNA for virus production and how much does the Core need?
Q: What forms do I need to fill out for recombinant virus production?
Q: What do I need to mark on the service request form?
Q: How much virus do I get?
shRNA tester samples – 5, 3 ml unconcentrated supernatant. Small scale production – 100 mls of supernatant concentrated 10X (10 mls of 10X virus).
Large scale production – 1 liter of supernatant concentrated 10X (100 mls of 10X virus).
Titers of unconcentrated lentivirus generally 1×106 transduced units/ml (1×107 transduced units/ml for 10X concentrated virus) with our standard rHIV vector using GFP as the readout. Titers may vary depending on the transgene being expressed, the system used, the quality of the proviral plasmid DNA and titering method.
Small scale production-VSVG env-concentrated – 100 mls of supernatant concentrated 10X (10 mls of 10X virus).
Large scale production-VSVG env-concentrated – liter of supernatant concentrated 10X (100 mls of 10X virus).
Small scale production-ecotropic or amphotropic env-unconcentrated – 100 mls of supernatant.
Large scale production-ecotropic or amphotropic env-unconcentrated – 1 liter of supernatant.
Titers are generally 1×107 transduced units/ml for VSVG enveloped, 10X concentrated virus and 1×106 transduced units/ml for ecotropic or amphotropic enveloped, unconcentrated virus with our standard rMMLV vector using GFP as the readout. Titers may vary depending on the transgene being expressed, the system used and the quality of the proviral plasmid DNA.
Adenovirus: The customer receives all virus that is produced. This amount varies dramatically depending on the viral system, transgene expressed, and quality of the starting virus. We aim for 4×1012 particles/ml. The typical yield is around 1.0-4.0×1013 total particles.
Q: How long does virus production take?
Adenovirus: Virus construction and transfection (code AT) and plaque amplification (code AA) – 4-5 weeks. Note: this process is the most difficult and most variable. It may take more than one attempt to make virus. This is in part because we overlay the cells and pick viral plaques for clonal isolation to assure the highest quality of virus. Adenovirus purification and expansion (code AL) – 2 weeks Titer (plaque assay) – 2-3 weeks
Lentivirus and Retrovirus: Two weeks once we receive sufficient quantity of the proviral plasmid to perform the transfection.
Q: What's the optimal concentration of virus that I should use for infection?
Q: Do I need a titer?
Q: How much media should I use during infection?
Q: Can serum be present in the media during viral infection?
Q: For adenovirus, what are the differences between viral particle (VP) and plaque formation unit (PFU)?
Q: What are the recommended storage conditions for the recombinant viruses?
Adenovirus Storage Buffer: The adenovirus is suspended in 10 mM Tris-HCl pH 7.4, 137 mM NaCl, 5 mM KCl, 1 mM MgCl2 to which glycerol has been added to 10% by volume.
Lentivirus and MMLV storage buffer: DMEM or as requested.
Q: What are RCAs?
Q: Which viral gene delivery system (adenovirus, retrovirus or lentivirus) is best to use for my experiments?
Lentivirus: Greater than 80% efficiency in most dividing cell types. Efficiency is lower in most non-dividing cell types and in most suspension cells. Lentivirus is a stable, low expression vector system due to integration into the host genome. Lentivirus may lead to mutation of genes in the host system.
Retrovirus (MMLV): gives <40% efficiency in most cell types, and requires active cell division. MMLV, like lentivirus, is a stable, low expression vector system due to integration into the host genome. MMLV may lead to mutation of genes in the host system.
|Viral Vector||Vector Core Storage Buffer||Temperature||Other|
|Adenovirus||The adenovirus is suspended in 10 mM Tris-HCl pH 7.4, 137 mM NaCl, 5 mM KCl,1 mM MgCl2 to which glycerol has been added to 10% by volume||-80° C||Avoid freeze/thaw|
|Lentivirus||Any||-80° C||Freeze-thaw a maximum of three times|
|MoMLV||Any||-80° C||Avoid freeze/thaw|
Viral Vector Comparison Chart
|Viral Vector||Maximum Insert Size||Preferential Target Cells||Common Research Applications||General Advantages|
|Adenovirus||7.5 kb||Transduces cells with CAR & avB integrins receptors very wellWill transiently transduce many primary cells at high efficiency||In vitro and in vivo transient transgene expression||Ease of use in vitro and in vivo High titers|
|Lentivirus||> 6.5 kb||Will transduce (via integration) differentiated non-dividing cells including most hematopoietic-derived cells and neurons, as well as dividing cells.||Stable integration allows for both production of permanent cell lines for in vitroapplications and long-term modifications for in vivoand ex vivoapplications||Transfects dividing and non-dividing cellsIntegration into genomeWide spectrum of target cells|
|Retrovirus (MoMLV)||> 6.5 kb||Transfects and integrates into wide variety of dividing cells||Stable integration allows for both production of permanent cell lines for in vitroapplications and long-term modifications for in vivoand ex vivoapplications||Integration into genomeWide spectrum of target cells|