Virus Vector Shared Resource Facility
Kristoffer Valerie, Ph.D.
Resource director
(804) 628-1004
Barbara Szomju
Resource manager
(804) 628-5336
The Virus Vector Shared Resource provides technical and consulting services for investigators who need virus vectors for basic laboratory and animal studies. Virus vectors for expressing proteins ectopically and knocking down gene expression are offered to Massey Cancer Center members and Virginia Commonwealth University investigators for costs well below commercial sources. The VVSR has the expertise, facilities, equipment and permission to safely produce and work with these types of viral vectors, reducing the risk for personnel working in Massey and VCU laboratories.
The facility does not operate under GMP-standards, and therefore cannot produce viral stocks for use in human clinical trials. The VVSR also develops new viral vectors based on need, and provides consulting services, training and education on the handling of recombinant viruses to interested research groups.
Facilities and equipment
The VVSR occupies dedicated, limited-access space in Massey Cancer Center and its adjacent Goodwin Research Laboratory. Equipment dedicated to VVSR includes:
- A Forma 6-ft. tissue culture hood.
- A Forma CO2 double-unit incubator.
- A Nikon tissue culture microscope.
Other molecular biology equipment and reagents (electrophoresis, microcentrifuge, DNA isolation) are provided. A Beckman ultracentrifuge with rotors is available for virus concentration and purification.
Services provided
Construction, purification and titering of viruses
Adenovirus
First-generation adenoviruses have the capacity to accommodate up to five kb DNA inserts under constitutive (CMV), inducible (Tet-On/Tet-Off), tissue- or tumor-specific promoters. In addition, adenovirus (AV) expressing shRNA for efficient, transient knockdown of specific gene expression is also offered. AV offers high efficiency of infection for a wide range of human and some rodent epithelial and endothelial (tumor and primary) cells with effects lasting up to 14 days or more in culture or in animals. It is an excellent system for expressing proteins in mammalian cells for therapeutic purposes, for animal studies or for isolation of large quantities of proteins from mammalian cells for functional studies. A set of plasmids with accompanying maps are available for convenient cloning and construction of the AV transfer plasmid.
Retrovirus
Retrovirus accommodates five to six kb DNA inserts that stably integrate into the genome of infected cells. The VVSR has vectors for drug selection (neo, puro, hygro), and GFP and DsRed for detection by fluorescence microscopy and isolation of cells by flow cytometry. The VVSR produces both mouse retrovirus and human LV that are pseudo-typed (have VSV-G on the surface instead of Env) that would infect virtually any kind of cell. Concentration of LV is possible, but large-scale virus production and purification is not generally offered by the VVSR. We also have the ability to make shRNA LV (constitutive or inducible). It is the ideal system for long-term sustained or inducible protein expression at relatively low levels. Engineered cells harboring LV can also be used for in vivo animal studies.
Lentivirus
Because of the versatility of the LV and superior properties (able to infect most mammalian cells from a variety of tissues without the need for cell growth) over the mouse retrovirus, most projects need or ask for LV vectors. We use a set of plasmids made by the Trono laboratory to make LV by transient transfection of HEK293T cells using three separate plasmids. Typically, we achieve titers >10E6 infectious units/ml that can be concentrated 100-fold by centrifugation. The VVSR has plasmids available that can produce a protein from an IRES-GFP/DsRed cassette, or short hairpin (sh) RNA for knocking down gene expression. In both cases, these viruses can be made so that protein expression (or knockdown) is constitutive or inducible. A set of plasmid with accompanying maps are available for convenient cloning and construction of LV.
Mouse retrovirus
A panel of mouse retrovirus plasmids is made available to the investigator. The VVSR has the pBabe series of plasmid vectors suitable for drug selection (hygromycin, puromycin, G418) of infected cells. The mouse retroviruses are produced by transient transfection of engineered HEK293-GP cells expressing inducible VSV-G. Viruses generated this way will infect proliferating mammalian cells from most species. Other amphotropic and ecotropic packaging cell lines are also available should a specific need by the investigator arise. Other mouse retroviruses expressing fluorescent proteins are also available. The procedure for making mouse retrovirus is basically the same as for making LV outlined above.
Consulting and support services
The VVSR will provide consultation on many aspects of virus vector technology, including experimental design, vector design and preparation of applications to the Institutional Biosafety Committee and IACUC for the use of virus vectors in laboratory and animal studies.
For laboratories that do not have prior experience working with virus vectors, Resource staff will provide instructions on how to safely perform experiments with virus vectors.
Service costs and access
To use these services, an application needs to be filled out and submitted to the VVSR. Approval to work with the virus is needed from the Institutional Biosafety Committee prior to work. At that time a few micrograms of plasmid DNA and a map are provided for cloning of DNA insert and consulting if necessary. After constructing plasmid with DNA insert, an appropriate amount of DNA is provided to the VVSR and virus will be made. Screening for expression, etc., is not done.
The VVSR asks investigators who use our services to acknowledge the VVSR and Massey Cancer Center in any publications that might result. Under the “Acknowledgment” or “Material and Methods” section, please state:
Virus was made by the Massey Cancer Center Virus Vector Shared Resource, Virginia Commonwealth University.
Costs for services offered by the VVSR are:
Service |
Cost |
1. Construction of new adenovirus (when provided with donor plasmid) |
$200 |
2. Purification of adenovirus (2 x CsCl banding, titering) |
$400 |
3. Titering of adenovirus by ELISA |
$100 |
4. Crude adenovirus prep (20 ml) and titering |
$150 |
5. Testing for replication-competent adenovirus (RCA) by qPCR |
$200 |
6. Generation of retrovirus supernatant and infection of cells |
$200 |
7. Titering of lentivirus by qPCR |
$200 |
8. Consulting/support services |
No charge |
Forms and surveys
