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Frequently Asked Questions: Custom AAV Service


Frequently Asked Questions: Custom AAV Service

  1. What's recombinant AAV (rAAV)?
  2. What's the biosafety requirement for using AAV?
  3. What's the cloning capacity for recombinant AAVs?
  4. Is recombinant AAV replication deficient?
  5. Is AAV stable? What is the recommended storage temperature?
  6. Can the AAV expression vector be used for transient transfection?
  7. Which serotype should I use in my experiment?
  8. What are the advantages of gene delivery by recombinant AAV (rAAV)?

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 .

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