VVCC services

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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 (1x1012-1014 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.