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Frequently Asked Questions   |   Storage Conditions   |   Comparison Chart
 

Frequently Asked Questions

Q: What is the required biosafety level for using recombinant viruses?
Q: What are RCAs?
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 long does virus production take?
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: How much virus do I get?
Q: What's the optimal concentration of viruses 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?
Q: Which viral gene delivery system (adenovirus, retrovirus or lentivirus) is best to use for my experiments?

Q: What is the required biosafety level for using recombinant viruses?
All viruses handled at the University of Michigan need to be registered with the UM Institutional Biosafety Committee (IBC). The IBC determines the biosafety level for each virus. In general, most of the recombinant viruses produced in the Vector Core are classified as Biosafety Level II for agents considered of ordinary potential harm. These recombinant viruses include E1, E3-deleted adenovirus and third-generation, self-inactivating (sin) lentivirus. Federal regulations can be found here.

Q: Will adenovirus/lentivirus/retrovirus work well in my cell models?
Both adenovirus and lentivirus have a wide host range in mammalian cell lines and primary cells. Both viruses infect dividing and non-dividing cells. There are few cell lines that cannot be infected with adenovirus or lentivirus. Retrovirus (MMLV) host range is not as wide as lentivirus, but still works well in many types of dividing cells. We recommend first testing your cell system with one of our adenovirus or lentivirus reporter viruses.

Q: How should I prepare DNA for virus production and how much does the Core need?
For adenovirus, we prefer 50 μg (minimum of 25 μg; preferably 1 μg/μl) of plasmid linearized with either PmeI, NheI, SfiI, or SwaI (see Protocols and Useful Links). We recommend that the DNA be heat inactivated after digestion and a sample run on the gel to confirm proper digestion. The DNA should then be phenol chloroform extracted, ethanol precipitated and resuspended in water. Preparation of the plasmid is important. We do not recommend gel extraction. For lentivirus and retrovirus, we need 100 μg and 840 μg of high quality plasmid for the small and large scale preps, respectively.

Q: What forms do I need to fill out for recombinant virus production?
Please see our Instructions and Forms page.

Q: What do I need to mark on the service request form?
The contact and billing information in the top box is required information. For ordering product, just write in the name of the product(s) in the appropriate box and the quantity that you request. Note: for UM customers, if you are ordering adenovirus construction, then code AA (plaque amplification) is required with code AT (DNA transfection for adenovirus construction). A large scale adenovirus purification and expansion does not include a titer (code AP).

Q: How much virus do I get?

Lentivirus:
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 1x10⁶ transduced units/ml (1x10⁷ 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.

Retrovirus (MMLV):
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 1x10⁷ transduced units/ml for VSVG enveloped, 10X concentrated virus and 1x10⁶ 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 4x10¹² particles/ml. The typical yield is around 1.0-4.0x10¹³ total particles.

Q: How long does virus production take?
Virus production is done first come, first served. The times listed below are estimates. The time may be longer due to large order volume or if a problem is encountered.

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?
The appropriate amount of virus used for infecting cells is very important for the outcome of your experiments. The optimal concentration differs dramatically between cell types. Too much virus uptake by the cells will cause cytotoxicity (with adenovirus) and too little virus will not give you 100% infection of the cells. The optimal concentration needs to be determined empirically. For these pilot studies, a marker virus such as β-gal or GFP is available. For adenovirus, optimal virus concentration for most cells is between 50 and 1000 MOI (multiplicity of infection; MOI x # of cells = pfu required). (click here for protocol). For lentivirus, optimal infection may require linker molecules or other "tricks" (click here for protocol).

Q: Do I need a titer?
A titer is the standard for determining how much virus to add to cells or inject into animals. Many journals will require a titer for publication. A titer (PFU/ml) is also needed to calculate MOI. Since the percent of live viral particles (VP) can vary drastically between any two viruses or preps, a titer is a way to normalize between viruses. The problem with titers is that they can also be variable depending on the type of assay used to determine the titer, the cells used and the person performing the assay. All Vector Core viruses are prepared the same way using standard procedures. There is very little variation in the VP:IU (infectious unit) ratio and the titers of all the viruses that we make are within a small range. Therefore, when using multiple viruses produced from the Vector Core, either particles or titer is fine.

Q: How much media should I use during infection?
We recommend the following amounts of virus-containing media for infection: 10-cm plate: 8-10 ml per plate; 6-well plate: 1 ml per well; 12-well plate: 0.5 ml per well; 24-well plate: 0.2 ml per well. This roughly reflects the surface area of each well or plate.

Q: Can serum be present in the media during viral infection?
Yes, as long as it's used with caution and it comes from a fetal animal. Serum from an animal that has been born runs a risk of having antibodies against the virus, particularly adenovirus.

Q: For adenovirus, what are the differences between viral particle (VP) and plaque formation unit (PFU)?
Viral particles (VPs) represent the total number of viral particles (live and dead). The amount of viable virus varies dramatically depending on how the virus is purified and stored. PFU (plaque formation unit) or infectious units represents the number of infectious or live viruses. It reflects the amount of working viruses in the preparation. VP/PFU ratio is generally 20:1 to 100:1. Vector Core adenovirus preps are about 25:1. Multiplicity of infection (MOI) is the number of viral copies per cell. It can also be thought of in terms of pfu per cell. It is calculated by MOI x # of cells= pfu required.

Q: What are the recommended storage conditions for the recombinant viruses?
We recommend that adenovirus be placed at -70°C or colder for long term storage. NOTE: Freeze-thawing will result in loss of infectious particles. For best results, thaw virus on ice and aliquot the amount you typically use per experiment into separate microfuge tubes. DO NOT freeze-thaw more than two times.
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?
Replication competent adenoviruses (RCAs) are wild-type like viruses in a population of replication-deficient viruses. RCAs result from a crossover event where the deleted E1 region of the virus re-inserts itself into the virus while being grown in packaging cells. Once this happens, the RCA adenovirus will replicate without the need of a packaging cell line and outgrow recombinant adenovirus in packaging cells. To avoid the occurrence of RCA, viruses should not be serially propagated. Recombinant adenovirus with high RCA can be rescued by clonal isolation after plaque assay.

Q: Which viral gene delivery system (adenovirus, retrovirus or lentivirus) is best to use for my experiments?
Adenovirus: 100% gene delivery efficiency in most cell types including dividing and non-dividing or primary cells. It's a high expression, transient vector system. There is no integration with the host system. Protocols involved in developing recombinant adenovirus are complicated. 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.

Storage Conditions

Comparison Chart