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 any project that involves "pushing 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. 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. AAV and lentiviral vectors are concentrated and purified by sucrose cushion ultracentrifugation, and viral titer is determined by qPCR. The typical yield is 200 uL of AAV (1x1013-1014 genocopies/mL) or lentiviral (1x107-108 particles/mL) vectors. 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.
Fee structure. The VVCC is non-profit entity, and we are able to keep our fees low for University of Minnesota 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. Investigators outside of the University of Minnesota who wish to use the VVCC for their custom cloning or viral packaging projects should contact the VVCC manager for information regarding external service fees.
Following consultation with the VVCC manager, the client receives a formal quote from the VVCC reflecting the unique features of the project. If the project requires multiple sub-cloning steps, significant investments in primers and plasmids, extensive sequencing, and/or reagent validation, 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.
Please note that we have unfortunately encountered some instances of problems with plasmids procured from commercial vendors, as well as materials provided by our clients. These problems can 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/or 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.
Timeline. The timeline for a custom cloning project depends on its complexity 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. Quantification of lentivirus titer via MOI requires an additional week. Procurement of relevant source DNAs and related Material Transfer Agreements is the responsibility of the client, and this can impact project cost and timeline.