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FAQ for adeno-associated virus (AAV)

What's the Biosafety requirement for using AAV

Recombinant AAV constructs, in which the transgene does not encode either a potentially tumorigenic gene product or a toxin molecule and are produced in the absence of a helper virus can be handled in Biosafety Level 1(BSL-1) facility. Otherwise it should be handled as biohazardous material under Biosafety Level 2 (BSL-2) containment. Please check with your Institutional Biosafety Committee or related NIH web site for detailed information, if you need more information.



Is recombinant AAV replication deficient?

For wild type AAV, replication is at extremely low efficiency, without the presence of helper virus, such as adenovirus. For recombinant adeno-associated virus produced these days, the replication and capsid genes are provided in trans (in pRep/Cap plasmid), and only the 2 ITRs of AAV genome is left and packaged into virion, while the adenovirus genes required are provided either provided by adenovirus or another plasmid, the likelihood for a recombinant AAV to replicate is theoretically impossible. This is in similar scheme to lentiviral vectors produced these days.



What¿s the cloning capacity for recombinant AAVs?

AAV has a packaging capacity of ~4.7Kb. Since the two ITRs of AAV is about 0.2-0.3Kb in total, the foreign DNA that could be introduced between these 2 ITRs should be <4.4Kb, which is much smaller than that of recombinant adenovirus (7.5Kb). In addition, when the length of inserted DNA between the 2 ITRs is close the maximal allowed, i.e., 4-4.4Kb, the packaging efficiency decreases significantly. For instance, for gene over-expression from cDNA, since the CMV-poly(A) element is about 1Kb, so the maximal allowable cDNA length is about 3Kb. In addition, if you are interested in GFP co-expression (from a separate expression cassette), given the additional CMV-EGFP-poly(A) is about 2 Kb, so the maximal cloning capacity for GFP co-expressing system is about 1.0-1.2Kb.

For double-stranded AAV (dsAAV), the capacity is only half of the single-stranded AAV (ssAAV).



What vector options do we provide for cDNA and shRNA, and what¿s their cloning capacity?

We have hundreds of different AAV ITR plasmids available for cloning, for gene over-expression and shRNA silencing knock down.

For over-expression AAV, you can choose from over 10 different promoters including ubiquitous promoter (CMV, CAG, CBA, EF1a) and tissue specific promoters (Synapsin, CamKIIa, GFAP, RPE, ALB, TBG, MBP, MCK, TNT, aMHC, etc), along with many different fluorescence reporter co-expression (GFP, RFP, mCherry, CFP, YFP, tdTomato, etc).

For shRNA silencing, you can choose from Pol III promoters (H1 or U6) and Pol II promoter (CMV) to drive the shRNA expression, or a Cre inducible shRNA expression.

Please click here for the AAV expression options.



Which AAV Serotype and dose(s) should be used for my study?

Though AAV(s) has been used for in vivo studies extensively, data regarding which serotype and dose are best for each application/tissue are not clear, and sometimes even controversial. This is probably due to the different experimental procedures used and end-point readout. To help investigators decide which AAV serotype and what dose should use his or her application, we have developed a AAV Serotype Testing Kit which contains AAV-GFP for several commonly used serotypes, so you can test different serotypes side by side in your own lab. It is advisable for investigators to do a small-scale, pilot study using these marker viruses.

A route to alter rAAV tropism exploits the natural capsid diversity of other serotypes, such as AAV1, and AAV3-6 etc, by packaging rAAV2 genomes into capsids derived from other AAV isolates. One approach employs hybrid trans-complementing constructs that encode rep from AAV2 whereas cap is derived from the other serotype displaying the cell tropism of choice.



How stable are AAV vectors? How should they be stored?

Stability studies carried out in house and by some colleagues show that purified AAV vectors are highly stable at temperatures of 4 C or less. We recommend aliquoting upon receipt and storing at -80C. Once an aliquot is thawed it can be stored at 4oC for short-term storage, e.g., 2-3 weeks, without significant loss of biological activity.



What's the difference between physical and genomic particles?

AAV genome particles relate to the viral particles that have been successfully packaged with the genome to be delivered. During the AAV packaging process many particles are formed lacking the genomic DNA, which lack the ability to transduce the cells they come into contact with and are therefore non-functional. As a researcher you probably want the concentration of functional AAV particles.



Want to learn more about AAV? read the introduction to AAV

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