Abstract
| Original language | English |
|---|---|
| Pages (from-to) | e3348 |
| Journal | Journal of visualized experiments : JoVE |
| Issue number | 57 |
| DOIs | |
| Publication status | Published - 2011 |
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Keywords
- Adenoviridae
- Genetic Vectors
- HEK293 Cells
- Humans
- Kidney
- Plasmids
- Transfection
- Virus Cultivation
Cite this
Production and titering of recombinant adeno-associated viral vectors. / McClure, Christina; Cole, Katy L H; Wulff, Peer; Klugmann, Matthias; Murray, Andrew J.
In: Journal of visualized experiments : JoVE, No. 57, 2011, p. e3348.Research output: Contribution to journal › Article
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TY - JOUR
T1 - Production and titering of recombinant adeno-associated viral vectors
AU - McClure, Christina
AU - Cole, Katy L H
AU - Wulff, Peer
AU - Klugmann, Matthias
AU - Murray, Andrew J
PY - 2011
Y1 - 2011
N2 - 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.
AB - 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.
KW - Adenoviridae
KW - Genetic Vectors
KW - HEK293 Cells
KW - Humans
KW - Kidney
KW - Plasmids
KW - Transfection
KW - Virus Cultivation
U2 - 10.3791/3348
DO - 10.3791/3348
M3 - Article
SP - e3348
JO - Journal of visualized experiments : JoVE
JF - Journal of visualized experiments : JoVE
SN - 1940-087X
IS - 57
ER -