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Frequently Asked Questions: AAV Service@BrainVTA

AAV FAQ

Frequently Asked Questions: AAV Service

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 ( Rep and cap genes encode viral replication and capsid proteins, respectively).  The gene of interest replace rep and cap and it is flanked by the ITRs which contain all the cis-acting elements necessary for replication and packaging.

2. What's the cloning capacity for rAAV?

AAV has a packaging capacity of 4.7Kb. If the GOI>4.7kb. We need to chose other vector systems.

3. Is recombinant AAV replication-deficient?

Yes, the rep and cap genes are provided in trans when producing the AAV particles, and only the ITRs sequences and the GOI are packaged. Additionally, the rep/cap plasmid and ITR plasmid do not share any regions of homology, which prevents the production of wild-type AAV.

4. Is AAV stable? What is the recommended storage temperature? 

For long-term storage, AAV should be stored at -80℃. However, if the virus is repeated freeze-and-thaw, it will cause a significant decrease of titer. For short-term storage, AAV is stable at 4°C for up to almost 1-2 weeks.

5. 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. But it will not be stable, it only used to confirm if the construction is working as intended.


6. Why can't I see the expression of GFP after AAV infection in my cell line? 

Poor expression in cells does not mean AAV cannot be expressed in vivo. AAV serotypes and promoters may affect the transduction ability of AAV particles in different cells. Thus, it is necessary to determine which serotype and promoter work best for your cell line.

Relative in vitro Infectivityof AAV Vectors:
Cell Line AAV-1 AAV-2 AAV-3 AAV-4 AAV-5 AAV-6 AAV-8 AAV-9 AAV-DJ AAV-DJ/8
Huh-7 13 100 2.5 0.0 0.1 10 0.7 0.0 500 0.2
HEK293 25 100 2.5 0.1 0.1 5 0.7 0.1 500 0.3
HeLa 3 100 2.0 0.1 6.7 1 0.2 0.1 667 0.2
HepG2 3 100 16.7 0.3 1.7 5 0.3 ND 1250 0.5
Hep1A 20 100 0.2 1.0 0.1 1 0.2 0.0 400 0.1
911 17 100 11 0.2 0.1 17 0.1 ND 500 0.0
CHO 100 100 14 1.4 333 50 10 1.0 25000 5.0
COS 33 100 33 3.3 5.0 14 2.0 0.5 500 0.3
MeWo 10 100 20 0.3 6.7 10 1.0 0.2 2857 1.0
NIH3T3 10 100 2.9 2.9 0.3 10 0.3 ND 500 0.1
A549 14 100 20 ND 0.5 10 0.5 0.1 1000 0.1
HT1180 20 100 10 0.1 0.3 33 0.5 0.1 333 0.2
Monocytes 1111 100 ND ND 125 1429 ND ND 100 ND
Immature DC 2500 100 ND ND 222 2857 ND ND 200 ND
Mature DC 2222 100 ND ND 333 3333 ND ND 100 ND
Note: Infectivity rates normalized to AAV-2 = 100. ND = Not Determined
1. Grimm, D. et al. (2008). J. Virol. 82: 5887-5911.

 

7. How can I choose the optimal serotype in my experiment?

More than 12 AAV serotypes are available for targeting different tissues and organs. Different AAV serotypes have different tissue tropism in vivo, for the common serotypes and their tropism, Please refer to the introduction to Adeno-Associated Virus (AAV). In addition, novel peptide-modified serotypes can also be produced on a case-by-case basis.

8. What are the advantages of gene delivery by rAAV?

rAAV has the capacity to produce a high titer virus with a broad spectrum of tropism in dividing and non-dividing cells and potential for long-term gene transfer with minimum immunogenicity.

9. How much plasmid do I need to provide and how to ship it, If I want to package an existing plasmid into a virus? 

You just need to drop mini-extracted plasmids on filter paper or PCR tube winded with sealing film and send it to us. Plasmid concentration≥100ng/ul, the total amount is 5-10ul. The filter paper should be labeled with a pencil.

10. What's the difference of AAV titer unit between GC/ml and vg/ml?

The AAV titer unit GC/ml and vg/ml can be used interchangeably.

11. What is the QC (quality control) method for testing AAV in BrainVTA?

Quality control include qPCR to quantify viral titers and silver stain to determine viral titer and purity after purification. If you need some special quality control, such as TEM, WB etc., we can also do it.

12. What is the difference for gene expression with AAV or lentiviral vectors in CNS?

Lentiviral particles don't spread well after injection into the brain because the particles are relatively large (90nm~100nm). LV also has a more toxic than AAV. In contrast, AAV particles spread more readily due to smaller size (20nm~100nm), Low immunogenicity, and can achieve specific expression. Lastly, pretreatment of the animal with mannitol about 15 min ahead of viral injection can aid the spread of viral particles in the brain as reported.

13. How much rAAV should be used for general animal experiments?

The amount rAAV used is highly dependent on the target tissue and route of administration. The following table summarizes the injection volume and injection methods of different tissues in mice. However, the exact dose should be determined empirically.
 
Tissue and organ Common injection method Recommended dose /vg Recommended volume
Most organs and blood systems Tail vein injection ≥1.0E+11 100-200ul
Heart, liver,kidney, pancreas, etc In situ injection 0.5-5.0E+10 1.5-3ul/site, Multisite injection
Heart, pancreas, muscle, etc Intraperitoneal injection 1.0-5.0E+11 50-100ul
Trachea, lungs Intratracheal injection 0.5-5.0E+11 50-100ul
Local blood vessels Vascular clip ~1.0E+10 ~100ul
Tumors Intratumor injection 0.5-5.0E+11 3-5ul/site, Multisite injection
Articular soft tissue Joint soft cavity injection 1.0-5.0E+10 5-10ul
CNS Stereotactic injection 1.0-10.0E+9 0.5-1.5ul/site,
Spinal cord In situ injection 1.0-10.0E+9 0.5ul/site, Multisite injection
Eye In situ injection ~5.0E+9 1.0~2.0ul

14. How long will AAV signal be observed after infecting mice?

The signal can be detected at 2-3 weeks, peaked at 6 weeks in mice, and about 8 weeks in rats. 

15. How long does the foreign gene express with AAV in mice?

  Half a year.

16. What if promoter specificity is bad?

The non-specific expression can be reduced by reducing the titer and the injection volume of the virus.

17. What if the promoter has a weak ability to drive the expression of genes?

The recombinant enzyme system (Cre) can be combined to amplify the gene effect.

18. What are the conditions for PHP.eB to cross the BBB?

PHP.eB has been shown to effectively cross the blood-brain barrier in C57 mice.

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If you have any questions about BrainVTA services, just email us at [email protected] or call us at 866-986-9598.