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1. What is recombinant AAV (rAAV)?
Recombinant AAV is the artificial adeno-associated virus vector which does not contain any AAV rep and cap genes which encode viral replication and capsid proteins, respectively. The rep and cap are replaced with a gene of interest which is flanked by the ITRs which contain all the cis-acting elements necessary for replication and packaging.
2. What's the biosafety requirement for using AAV?
If the transgene which inserts into the recombinant AAV vector dose not encode either a potentially tumorigenic gene product or a toxin molecule, the use of AAV can be handled in Biosafety LevelI (BSL-1)facility. Otherwise it should be handled as biohazardous material under Biosafety LevelII (BSL-2) containment.
3. What's the cloning capacity for recombinant AAVs?
AAV has a packaging capacity of 4.7Kb. As the two ITRs of AAV is about 0.3 Kb, the transgene that could be insert into the AAV vector should be < 4.4kb.
4. Is recombinant AAV replication deficient?
Yes, the replication and capsid genes (in rep/cap plasmid) are provided in trans when the AAV is produced, and only the ITRs sequences and the transgene are packaged into virion. Additionally, the rep/cap plasmid and cis plasmid do not share any regions of homology, which prevents the production of wild-type AAV through recombination system.
5. Is AAV stable? What is the recommended storage temperature?
For long-term storage, AAV should be stored at -80?C. However if the virus is repeated freeze-and-thaw, it will cause significant decrease of titer. For short-term storage, AAV is stable at 4?C for up to almost 2-3 weeks.
6. Can the AAV expression vector be used for transient transfection?
The AAV expression vector can be transfected into cells without the packaging vectors to measure gene expression. This will be a transient transfection and therefore will not be stable, but will allow you to confirm that your construction is working as intended.
7. Which serotype should I use in my experiment?
AAV is an excellent tool which has been used for in vivo studies. However different applications/tissues studies have to choose different serotypes packaging AAV. And data regarding which serotype is best for each application/tissue is not clear, and sometimes even controversial. This is probably due to the different experimental procedures used and end-point readout.
Below are some preliminary guidelines.
AAV1: CNS, Eye, Heart, Lung, Skeletal muscle
AAV2: CNS, Eye
AAV5: CNS, Eye, Lung
AAV6: Adipose, Heart, Liver, Lung, Skeletal muscle
AAV8: Adipose, CNS, Eye, Liver, Skeletal muscle;
AAV9: Adipose, CNS, Eye, Heart, Liver, Lung, Skeletal muscle
8. What are the advantages of gene delivery by recombinant AAV (rAAV)?
rAAV is the most promising tool for gene delivery owing to its non-integrating nature and minimum immnunogenicity. rAAV also has the potential for long-term gene transfer and capacity to produce high titer virus with broad spectrum of tropism in dividing and non-dividing cells .