Production and Titering of Recombinant Adeno-associated Viral Vectors

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 replicationdeficient. 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.

In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9

“…High titer AAV1/2 particles were produced using AAV1 and AAV2 serotype plasmids at equal ratios and pDF6 helper plasmid in HEK293FT and purified on heparin affinity column 34 . Titering of viral particles was done with qPCR.…”

Medial Amygdala <b><i>Kiss1</i></b> Neurons Mediate Female Pheromone Stimulation of Luteinizing Hormone in Male Mice

“…A small incision was made to expose the skull and a small craniotomy was drilled directly above each target site. Each DREADD vector plasmid was packaged into rAAVs (serotype 1/2) using the method described [23]. One construct (pAAV-EF1a-DIO-hM3D[Gq]-mCherry [Addgene plasmid #50460], a gift from Bryan Roth) encodes a modified G protein-coupled receptor hM3D(Gq) that, after binding to its non-endogenous ligand, clozapine N-oxide (CNO), induces neuronal burst firing [24, 25].…”

Pharmacology of Heparin and Related Drugs

“…It binds heparin and can be isolated using heparin affinity chromatography (McClure et al, 2011). Electron microscopy has been used to study the capsid-heparin complex in two studies published almost simultaneously (Levy et al, 2009;O'Donnell et al, 2009).…”