UNIT 14D.1 Production and Discovery of Novel Recombinant Adeno-Associated Viral Vectors
Published Online: 1 AUG 2012
Copyright © 2012 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Microbiology
How to Cite
Mueller, C., Ratner, D., Zhong, L., Esteves-Sena, M. and Gao, G. 2012. Production and Discovery of Novel Recombinant Adeno-Associated Viral Vectors. Current Protocols in Microbiology. 26:D:14D.1:14D.1.1–14D.1.21.
- Published Online: 1 AUG 2012
In this unit, we describe the detailed procedure for a three-plasmid transfection method for rAAV production, and discuss its advantages, limitations, and troubleshooting techniques. We further discuss the rAAV purification process using CsCl gradients, as well as subsequent quality control methods using SDS-PAGE and real-time PCR to assess vector purity, packaging efficiency, and viral titer. Finally, we elaborate on a PCR-based strategy that can be used to discover novel AAV capsid sequences from primate tissue, which can be used to develop newer-generation rAAVs with a greater diversity of tissue tropism for clinical gene therapy. Curr. Protoc. Microbiol. 26:14D.1.1-14D.1.21. © 2012 by John Wiley & Sons, Inc.
- AAV production;
- AAV packaging;
- cesium chloride