1. What is recombinant AAV (rAAV)?
2. What's the cloning capacity for rAAV?
3. Is recombinant AAV replication deficient?
4. Is AAV stable? What is the recommended storage temperature?
5. Can the AAV expression vector be used for transient transfection?
6. Why can't I see the expression of GFP after AAV infection in my cell line?
7.How can I choose the optimal serotype in my experiment?
8.What are the advantages of gene delivery by rAAV?
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?
10.What's the difference of AAV titer unit between GC/ml and vg/ml?
11.What is the QC (quality control) method for testing AAV in BrainVTA?
12.What is the difference for gene expression with AAV or lentiviral vectors in CNS
13.How much rAAV should be used for general animal experiments?
14.How long will AAV signal be observed after infecting in mice?
15.How long does the foreign gene express with AAV in mice?
16.What if promoter specificity is bad?
17.What if the promoter has a weak ability to drive the express of genes?
18.What are the conditions for PHP.eB to cross the BBB?

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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 ( 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 system.
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 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 works best for your cell line.
Relative in vitro Infectivity¹ of 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 high titer virus with broad spectrum of tropism in dividing and non-dividing cells and potential for long-term gene transfer with minimum immnunogenicity.
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 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 brain because the particles are relatively large (90nm~100nm). LV also have 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 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 volum
Most organs and blood systems	Tail vein injection	≥1.0E+11	100-200ul
Heart,liver,kidney,pancreas, ect	In situ injection	0.5-5.0E+10	1.5-3ul/site, Multisite injection
Heart, pancreas,muscle, ect	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 in 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?
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 express 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.