Welcome to the VVCC
Viral Vector and Cloning Core
Overview
The University of Minnesota Viral Vector and Cloning Core (VVCC) was established in May 2017 to help meet the needs of investigators requiring DNA and viral vector tools for their research programs. Since then, we have completed 400+ custom cloning and 1000+ viral packaging projects for 70+ different clients across the University of Minnesota Medical School, College of Pharmacy, School of Dentistry, College of Veterinary Medicine, College of Science and Engineering, and College of Biological Sciences. Our fees, particularly for custom projects, are well below those you will find with external service providers. In addition, the in-person service offered by the VVCC is difficult to replicate by an external provider.
Background
DNA constructs and viral vectors are used in biomedical research to enhance or suppress gene expression, drive expression of foreign genes in defined cell types, and introduce or correct genetic mutations in cells over a range of developmental stages. These tools can be exploited to probe the molecular basis of organ function and dysfunction, the molecules and neural circuitry underlying normal or pathological behavior, as part of gene therapy approaches, to generate new animal disease models, and to facilitate high-throughput screening efforts that may yield new compounds for treatment of medical problems.
The generation of new DNA constructs and viral vectors begins with techniques often referred to as “cloning” or “sub-cloning” - an array of molecular biology procedures that permit the strategic cutting and pasting of DNA segments together or the introduction of foreign sequences or defined mutations into coding sequences of interest. Viral packaging requires additional techniques in cell culture and manipulation. Sub-cloning and viral packaging procedures require specific lab infrastructure and expertise. Not all labs are willing or able to make the relevant investments needed to conduct this type of work, thus generating a need for service that can restrict the scope of research that a group can pursue, or that needs to be filled by collaborating with another research group or engaging an external service provider.
Contact Us
VVCC Director
Ezequiel Marron, PhD
[email protected]
Services
Custom cloning. The VVCC will design and create custom DNA constructs that can be used for a variety of purposes, including expression of cDNAs of interest in mammalian or bacterial systems, suppression of gene expression, and/or as precursors (shuttle vectors) for subsequent viral packaging efforts. Examples of custom cloning projects completed by the VVCC include epitope tagging, introduction of loxP sites for Cre-dependent gene expression, sub-cloning a cDNA of interest downstream from a cell-specific promoter, introduction of point mutations or deletions into cDNAs of interest, and design and production of custom shRNA or CRISPR/Cas cassettes. Clients interested in generating custom shuttle vectors for AAV or lentiviral packaging projects should keep in mind that recombinant vectors can accommodate a genome less than or equal to that of the wild-type virus from which they were derived. For AAV vectors, the upper limit for foreign DNA sequence is 4.7 Kb. For lentiviral vectors, the upper limit is closer to 9 Kb. The VVCC reserves the right to decline projects that "push the limits" in this regard.
Viral packaging. The client provides or recruits the VVCC to create a shuttle vector of suitable quality and quantity for packaging in an AAV or lentivirus (2nd or 3rd generation) format. AAV vectors are concentrated and purified by sucrose cushion ultracentrifugation, and viral titer is determined by qPCR. The typical yield is 200 uL of AAV (1x1012-1013 genocopies/mL). Vectors produced have proven to be of sufficient quality and purity for use in the most sensitive in vitro (e.g., primary cell cultures) or in vivo (e.g., intracranial manipulations) applications.
IBC Requirements
University of Minnesota investigators will require Institutional Biosafety Committee (IBC) approval to work with reagents produced by the VVCC. It is important to submit an IBC protocol, or an IBC protocol amendment, as soon as possible once committing to seeking VVCC support for your efforts. To facilitate this process, we have prepared documents that will help you secure IBC permission for work with recombinant DNA and/or viral vectors. The documents are somewhere.
FAQs
Frequently Asked Questions
How much will my project cost? The VVCC is non-profit entity, and we are able to keep our fees low for UMN investigators due to the generous support of institutional partners including the Department of Pharmacology, Medical Discovery Team on Addiction, MnDRIVE, Institute for Translational Neuroscience, and the Masonic Cancer Center. For UMN investigators, custom cloning projects start at $495 (scaling upwards with complexity) and AAV viral packaging projects start at $445 (or $545 if a plasmid maxi-prep is required). External Investigators should contact the VVCC director for information regarding external service fees. Following consultation with the VVCC director, the client receives a formal quote from the VVCC reflecting the unique features of the project.
What source material will the VVCC accept for a custom cloning or packaging project?Sequence-validated plasmid DNA is the only acceptable starting material for VVCC-related projects. We will not accept cells or cell extracts, mRNA samples, PCR products, or other non-plasmid RNA or DNA materials as templates for custom cloning projects. We have unfortunately encountered some instances of problems with plasmids procured from commercial vendors, as well as materials provided by our clients. These problems result in time- and reagent-consuming trouble-shooting, or may not be realized until after vectors are prepared. As such, packaging or cloning projects involving materials obtained from external sources will require pre-validation using diagnostic restriction enzyme tests and sequencing before any work will proceed. Similarly, source materials provided by the client must be accompanied by complete DNA sequence information, allowing the VVCC to conduct limited diagnostic restriction enzyme testing. Any costs related to pre-validation of source materials will be included in the service quote provided to the client prior to project initiation. Please note that procurement of relevant source DNAs and related Material Transfer Agreements is the responsibility of the client.
How long will it take to complete my project? The timeline for a custom cloning project depends on its complexity, the quality of source DNA provided, and our current workload. While a project involving 1 sub-cloning step will generally take 2-4 weeks to complete once all source DNAs are in-hand, sub-cloning can be unpredictable and we appreciate your patience if and when setbacks occur. Most AAV and lentiviral packaging projects can be produced within 3-4 weeks after receipt of the items required to initiate virus production. If a project requires multiple sub-cloning steps, significant investments in primers and plasmids, extensive sequencing, and/or reagent validation, timelines and rates will scale accordingly. The VVCC reserves the right to decline to pursue a project if it is overly complex or likely to require extensive trouble-shooting.
What AAV serotypes are available to my project? We have completed AAV packaging projects involving AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVDJ, AAVPHP.eb, AAVrh10, and AAV2retro serotypes, and are happy to work with you to determine the optimal serotype for your needs.
How do I acknowledge support from the VVCC in a manuscript, poster, or oral presentation? We ask you to consider including VVCC staff as co-authors if they provide substantial intellectual input to the study. In all cases, include the following statement (or suitable alternative): All viral vectors used in this study were generated by the University of Minnesota Viral Vector and Cloning Core (Minneapolis, MN).
Will the VVCC provide a letter of support for my grant application? Absolutely! Please send requests for letters of support to [email protected]; we will work with you to develop a project-specific letter that can be included with your grant application.
How should I store my viral vector? AAV vectors are stable if stored at -80oC. Viruses with a lipid membrane (e.g., lentiviruses), however, can experience a ~10-fold reduction in titer for every 6 months of storage at -80oC. Accordingly, the VVCC recommends that lentiviral vectors are used within 2-3 months of production unless the titer far exceeds what is required for the project. In addition, it may be better to order smaller lots of virus repeatedly rather than one large lot.
Why does viral titer vary from project-to-project? Project-specific shuttle vectors are the main variable in packaging projects. When viral production fails or titer is low, therefore, the problem can typically be traced back to the shuttle vector. Expression of genes in virus-producing cells may inhibit viral production as this process depends on host cellular infrastructure and overall health. Typically, highest titers are obtained when vectors encode “innocuous” genes or when the promoter is weak in virus-producing cells.