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.
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.
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
The costs (effective July 1, 2013) for different size viral vector preparations and titering. We offer a 20% discount on viral preparations and Taqman titer analyses for our academic customers. Please feel free to inquire about the details of our pricing structure. Once we know the amount of virus you would like we can provide you with an estimated date of delivery. Please note, the actual date for the preparation will be scheduled once we have received the plasmid and preferably a plasmid map.
Larger volume preparations are available upon request. Please contact us for further consultation regarding VCM volume, turnaround time and prices.
|Preparation Size||ml VCM Produced||Cost||Academic Cost||Volume Aliquotes|
|1 Plate||0.43 ml||$631||$358||4-100μl, 1-30μl|
|4 Plates||1.78 ml||$1,214||$690||2- 500µl, 1-250µl, 5-100µl, 1-30µl|
|6 Plates||2.58 ml||$1,517||$853||4- 500µl, 5- 100µl, 1-30µl|
|12 Plates||5.16 ml||$2,217||$1,260||2-1ml, 5-500µl, 2-250µl, 1-100µl|
|24 Plates||10.32 ml||$3,560||$2,023||1-5ml, 3-1ml, 3-500µl, 2-250µl, 3-100µl|
The smallest aliquote may vary by several microliters due to fluid loss.
Titration by fluorphore: $88
Each additional vector preparation made in parallel: $69
See the Taqman Titer chart for Titration by real-time PCR
Charges for Additional Services
|1 vector||2 vector||3 vector||4 vector|
Location: Thomas Building, D1-324
Fred Hutchinson Cancer Center
P.O. Box 19024
Mail Stop: D1-100
Seattle, WA 98109-1024