Title
Production and discovery of novel recombinant adeno-associated viral vectors
UMMS Affiliation
Gene Therapy Center; Department of Pediatrics; Department of Medicine; Department of Neurology; Department of Microbiology and Physiology Systems
Date
8-1-2012
Document Type
Book Chapter
Medical Subject Headings
Dependovirus; Genetic Vectors; Gene Therapy
Disciplines
Allergy and Immunology | Biomedical Engineering and Bioengineering | Genetics and Genomics | Medical Genetics | Pediatrics | Respiratory Tract Diseases
Abstract
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.
Comments
Citation: Curr Protoc Microbiol. 2012 Aug;Chapter 14:Unit14D.1. DOI: 10.1002/9780471729259.mc14d01s26