The development of retroviral delivery systems for mammalian cells has greatly enhanced the ability of scientists to deliver genetic material to cells of diverse origin in order to study the role of a particular protein or network of proteins in a specific cell type or biological process. The goal of the retrovirus production unit is to assist Cancer Center members with the preparation of high-titer retrovirus (e.g. lentivirus). The centralization of such services provides economy of scale, high-throughput production, increased biosafety, and quality control in the virus preparation process. The Facility provides a variety of services including prepackaged vector preparations, custom vector packaging, and vector titrations.
The Protein Expression Facility’s work with recombinant retrovirus DNA is registered with the Wistar Institute IBC (approval #: 21102382, exp. 3/2014)
1. Fluorescent protein tracer: These vectors express fluorescent proteins (e.g. GFP, DsRed2, mCherry) from a strong constitutive promoter (e.g. CMV, EF1a, PGK) and are useful for marking cell populations and to experimentally determine the transduction efficiency of a cell line.
2. Luciferase vector: These vectors express firefly luciferase from a strong constitutive promoter (e.g. EF1) and are useful for monitoring cell populations in vitro and in vivo.
3. pLKO shRNA controls: This vectors serve as controls (e.g., empty, non-targeting, siGFP, siLuc, etc) for experiments using TRC (pLKO.1) shRNA plasmids.
* All prepackaged vector preparations are VSVg pseudotyped, non-replicating lentivirus and come as filtered, concentrated vector in PBS. Concentration is performed by ultracentrifugation. All vectors, except pLKO-based, are quantitatively titrated using HEK293T cells and percentage of transduced cells determined by flow cytometry. Note: pLKO.1 vectors do not have a fluorescent marker. Therefore, the titer of these vector stocks has been determined by a p24 assay.
** The Wistar Institute Vector production unit has purchased the above plasmids from Sigma-Aldrich and Open Biosystems and does not profit from the sale of lentivirus in any way. All lentiviral vectors produced by the Wistar Vector Production Unit and distributed to Cancer Center laboratories are for use in research only.
View a list of available vectors.
The facility will prepare non-replicating, VSVg pseudotyped vectors by:
• Transfecting retrovirus vector, 2nd generation packaging plasmid, and VSVg plasmid into HEK293T cells
• Collect viral supernatants at 12-24 hr intervals
Retrovirus production services are ordered based on volume of conditioned cell culture supernatant.
30 ml conditioned cell supernatant: 20ug
60 ml conditioned cell supernatant: 40 ug
120 ml conditioned cell supernatant: 80 ug
B. 30ml conditioned cell supernatant
C. 60ml conditioned cell supernatant
D. 120ml conditioned cell supernatant
E. 30ml concentrated supernatant resuspended in PBS
F. 60ml concentrated supernatant resuspended in PBS
G. 120ml concentrated supernatant resuspended in PBS
The Retrovirus Production unit will concentrate (~1000X) vector preparations by ultracentrifugation. All concentrated vectors are provided in PBS as 5-10 ul aliquots.
Vector production unit determines titers of vectors using a quantitative titration protocol on HEK293T cells, if the vector contains a fluorescent marker. Please do NOT request titrations on other cell lines or using alternative methods.
Guidelines for custom lentivirus preparation:
1. To schedule custom preparation of lentivirus, investigators must submit a completed plasmid submission form.
2. Customer billing information and a valid grant number (Wistar investigators only) or PO# (all external users) must be provided prior to the initiation of any work.
3. To address safety concerns, please provide information on the nature of your vector (i.e. promoter, markers, non-replicating) and insert (i.e. select agent, oncogene, immunosuppressant, toxin, etc.).
4. Please provide the appropriate amount of transfection quality (i.e. Qiagen, Wizard prep, etc.) lentiviral vector DNA, containing your gene of interest, which you want to have packaged into the virus, with supporting submission form. Note: We do not accept DNA minipreps.
5. Please indicate the concentration of your transfer vector (we prefer a working concentration of 0.5-1μg/μL).
6. Please indicate the services (volume of cell supernatant, concentration, titration) you are requesting.
7. Due to variability in packaging of different retroviral vectors, production of a minimum titer cannot be guaranteed. Whenever possible, newly produced vectors should be titrated.
8. It is the responsibility of the investigator to register the intended specific use of recombinant DAN technology (i.e. retrovirus use) with Wistar’s Institutional Biosafety Committee (IBC) as required by the most current NIH Guidelines for Research Involving Recombinant DNA Molecules. For a full description of requirements, please consult the NIH Guidelines.
9. Please acknowledge the Protein Expression Facility if viruses are used in the generation of publication data.