Studies in this application require AAV or lenti-viral vectors from plasmids that the individual researches design using standard recombinant DMA techniques. The goal of the Vector Core Facility is therefore the production of research grade AAV and lenti-viral vectors for pre-clinical studies is proposed in these projects. This function will ensure the availability and quality of recombinant AAV and lentiviral vectors for the different projects that require delivery vectors for expression of therapeutic transgenes. AAV vector is produced in a helper virus-free system based on large-scale plasmid transfection of HEK-293 cells using two helper plasmids that supply AAV rep/cap and adenoviral helper functions and a third plasmid encoding the recombinant vector. This will allow production of a variety of different vectors for the investigators. The lentiviral vectors will be produced in 293T cell line by transfection. The lenti-viral vectors will be concentrated by either ultracentrifugation or ultra filtration. Packaging envelops available include VSV-G, Mokola, Rabies, MLV-Ampho, MLV-10A1, LCMV-WE, and LCMV-Arm53b. The Facility will provide reproducible yield and purity of vector, and will be more cost-effective than vector production in individual laboratories, in particular for experiments that involve large animal models. The service of the Core will include large-scale preparation of helper and vector plasmids, large-scale transfection of HEK-293 and 293T cells using calcium phosphate precipitation, recovery and purification of recombinant AAV by cell lysis followed by column chromatography or gradient centrifugation, dialysis, sterile filtration, and storage of purified vector, quantitative slot blot hybridization, lentiviral concentration and characterization. The Core will perform additional tests on purity and sterility of vector and assist the researchers that requested the vectors with functional assays. Standard AAV preparations are expected to yield about 10[13] vector genomes, while scale up will result in production of about 10[14] vector genomes per preparation. Scale-up of vector production using a roller bottle method has been optimized (Biotechniques 34:1, 2003) and will be applied to production of vector for non-human primate studies. The yield of lentiviral vectors is the range of 1x10[9] infectious units/ml, which may vary depending on the pseudotype envelopes.
Showing the most recent 10 out of 90 publications
