AAV production and packaging
Working in conjunction with the University of Minnesota Viral Vector and Cloning Core (VVCC), the VIC supports the generation of AAV vectors – including custom vectors – for members of the UMN addiction research community. Providing this labor-intensive service through a centralized entity with skilled staff represents a critical efficiency for the VIC user base, and facilitates the centralized examination, evaluation, and interpretation of data from a large and broad array of vector tools. Tools generated with support from the VIC are expected to promote engagement with the other three Cores, creating synergies that will strengthen the impact and innovation of all supported projects, and enhance the scope and impact of UMN research in the area of addiction.
Projects undertaken by the VIC could include the packaging of stock/commercial AAV vectors including those that express genetically encoded fluorescent proteins (e.g., GFP, mCherry) and/or neuromodulatory tools (opsins, DREADDs) under the control of cell-specific promoters (e.g., hSyn, CaMKIIα). These and other vectors can be packaged in a serotype (e.g., AAV1, 2, 3, 4 5, 6, 7, 8, 9, rh10, AAV2retro, DJ, PHP.B, PHPeB, and PHP.S) suitable for the cell population under investigation. AAV vectors are concentrated and purified by sucrose cushion ultracentrifugation, and viral titer is determined by digital PCR. The typical yield is 200 uL of AAV vector (>1x1013 genocopies/mL). Vectors produced by the VIC 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.
The VIC will also support the development of custom reagents to fill project-specific needs, including vectors harboring gene-specific shRNA or guide RNAs, or cDNAs downstream from a promoter of interest. Cre-dependent (DIO and DO) versions of AAV vectors are popular among VIC clients, as they can be used in conjunction with an expanding array of Cre transgenic mouse and rat lines that have revolutionized circuit-based neuroscience investigations over the last decade. VIC staff will design and produce the DNA plasmids required for custom AAV production.