Gammaretrovirus, Lentivirus, and Foamy Virus Vector Production

Location: Thomas Building, D1-171
Contact phone: (206) 667-4425
Contact fax: (206) 667-6124
Contact e-mail: hkiem@fredhutch.org

Our service include assisting investigators with design, construction and production of viral vectors for the introduction of gene expression cassettes or gene silencing constructs into cells of interest. For lentiviral packaging, our average yield for viral preps is in the range of 1 x 108 infectious units (IU)/mL.

Procedure

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 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, 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 services rendered if the following conditions apply:

  1.  Your transfer vector was not designed by the Vector Core Facility
  2.  The viral prep fails to yield a satisfactory titer
  3.  The control construct yields a titer in excess of 1 x 108 TU/mL

An outline of our protocol can be found at the Kiem lab web site.

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.