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
Publication Date
2012-08-01
Document Type
Book Chapter
Subjects
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.
DOI of Published Version
10.1002/9780471729259.mc14d01s26
Source
Curr Protoc Microbiol. 2012 Aug;Chapter 14:Unit14D.1. DOI: 10.1002/9780471729259.mc14d01s26
Journal/Book/Conference Title
Current Protocols in Microbiology
Related Resources
PubMed ID
22875565
Repository Citation
Mueller, Christian; Zhong, Li; Ratner, Dmitry; Sena-Esteves, Miguel; and Gao, Guangping, "Production and discovery of novel recombinant adeno-associated viral vectors" (2012). Pulmonary and Allergy. 72.
https://escholarship.umassmed.edu/peds_pulmonary/72