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

Related Resources

Link to article in PubMed