Production and titering of recombinant adeno-associated viral vectors

J Vis Exp. 2011 Nov 27;(57):e3348. doi: 10.3791/3348.

Abstract

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Publication types

  • Video-Audio Media

MeSH terms

  • Adenoviridae / classification
  • Adenoviridae / genetics*
  • Adenoviridae / isolation & purification*
  • Genetic Vectors / classification
  • Genetic Vectors / genetics*
  • Genetic Vectors / isolation & purification*
  • HEK293 Cells
  • Humans
  • Kidney / cytology
  • Plasmids / genetics
  • Transfection / methods
  • Virus Cultivation / methods*