Vector Production
Creating High-Quality and Effective Viral Vectors
As a lab within the Cooperative Center of Excellence in Hematology (CCEH) at Fred Hutch, the Vector Production core assists investigators with the design, construction, and/or production of viral vectors (gammaretrovirus, lentivirus, foamy and adeno-associated virus) for introducing select genes of interest or gene silencing constructs into cells of interest.
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Lentivirus
Lentiviral vectors, derived from the single-stranded RNA retrovirus HIV-1, have been used extensively as gene transfer tools. LV vectors are unique in their ability to infect nondividing cells, efficiently integrate into the host genome, carry large transgenes, and allow for stable long-term transgene expression. The lentiviral vector only contains the LTRs and the packaging signal, Ψ. Lentiviral packaging genes are provided on separate plasmids, so the pseudo lentiviral particles are replication deficient.
Despite their advantages, however, LV vectors are associated with a relatively high risk of insertional mutagenesis as they integrate into the host genome, thus potentially retaining the ability to induce oncogenesis.
Adeno-Associated Virus (AAV)
AAV is a single-stranded DNA parvovirus that consists of a small, single-stranded DNA. AAV deliver genetic material that nests in the cell nucleus and does not permanently integrate into the cell DNA. AAVs have a smaller genetic packaging capability compared to other viral vectors. These viruses are preferred over lentiviruses because they remain primarily episomal, while lentiviruses integrate into the genome. Also, AAV produces the lowest immune response, is non-pathogenic even in the wild-type state, and is thus thought to be the most suitable virus for therapeutic applications.
Gammaretrovirus
Gamma retroviral vectors are derived from MLV (Murine Leukemia Virus). In contrast to LV, gamma retro virus cannot infect non-dividing cells. Similar to LV, gamma retrovirus mediate stable integration into the host genome. Interestingly, gammaretroviral and lentiviral vectors differ in regard to preferred integration sites, with integration of gammaretroviral vectors more frequently in transcription start sites and lentiviral vectors in active genes. Limitations of gammaretroviral vectors arise from the poor infection of non-dividing cells, faulty reverse transcription, intracellular restriction factors and the risk of insertional mutagenesis.
Foamy Virus
Foamy viruses (FVs) are nonpathogenic retroviruses that infect various animals, and can be transmitted to humans through zoonotic infection. Due to their non-pathogenic nature, broad tissue tropism and relatively safe integration profile, FVs have been engineered as novel vectors (foamy virus vector, FVV) for stable gene transfer into different cells and tissues. FVVs have emerged as an alternative platform to contemporary viral vectors.
The investigator constructs the transfer vector containing their gene of interest. The vector packaging facility has cloning vectors (containing the GFP marker) that can be used to make these constructs. See the cloning vectors available and contact us to receive the DNA. (If you would like the facility to construct your transfer vector, contact us.)
Investigators can purify their plasmid transfer vector and provide to the facility at a concentration of 0.5 to 2 µg/µl. We require at least 30 µg of DNA per plate equivalent. Please note that all standard plasmid preps should be cleaned with phenol and chloroform before submitting DNA. The most common cause of failure for vector preps are insufficiently cleaned transfer vector preps. In order to circumvent this issue, the facility can amplify the DNA for you. (We strongly recommend that for each plasmid preparation you provide to the Core that you perform several diagnostic restriction digests to confirm that the plasmid preparation is the expected one.)
The facility provides the helper plasmids for the virus preparation which includes the viral VSV-G envelope in case of lentiviral preparations. We offer not only the standard second but also third generation packaging for lentiviral preparations upon special request.
For routine preparations, we use concentrated virus containing media (VCM) frozen in convenient aliquots (see price list). If your transfer vector contains a fluorescent tag, we can perform a titer analysis by flow cytometry for you. If your transfer vector does not contain a fluorescent tag, our facility is still able to provide you with a reliable titer via real-time PCR analysis (Taqman). Please allow additional two weeks for this procedure as transduced cells must be passaged prior to analysis.
For quality assurance purposes, we will prepare a viral preparation with an appropriate control vector in parallel as a reference and to guard against procedural failure. Please be aware that we will have to charge for the viral preps that fail to yield a satisfactory titer if our control construct yields a titer equal to or greater than 1x108 IU/ml.
Constructs Available to Our Customers
In 2011, we began offering standard cloning vectors for gene expression. These are based on our reference vector and allow for the inclusion of cDNA or the exchange of the fluorescent marker. These vectors are available for our customers only. We do not consent to third party distribution or to use that does not involve our vector services. Laboratories in violation of this arrangement will also not be eligible for future services.
IRES = internal ribosome entry site (bicistronic expression)
CAR = cis acting region
W = WPRE
Getting Your Virus Preparation Started
Please fill out the order form, and include the following information.
- Is the plasmid for foamy viral, gammaretroviral or lentiviral preparation?
- How many plates would you like prepared (1, 4, 6, 12, or 24)?
- Do you wish titration via fluorescent reporter gene expression (in case your transfer vector expresses a fluorophore like GFP) or Taqman PCR?
If a transfer vector is provided, please provide the DNA concentration for us. Ideally we prefer a concentration of 0.5 to 2 µg/µl. We require at least 30µg of DNA per plate equivalent. Please note that all standard plasmid preps should be cleaned with phenol and chloroform before submitting DNA. The most common cause of failure for vector preps are insufficiently cleaned transfer vector preps. In order to circumvent this issue, we prefer to amplify the DNA for you.
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Megha Gupta, Ph.D.
Hans-Peter Kiem, M.D., Ph.D.
Location / Address
Location: Thomas Building, D1-324
Mailing Address:
Fred Hutchinson Cancer Center
P.O. Box 19024
Mail Stop: D1-100
Seattle, WA 98109-1024