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Construction and Packaging Service of virus Vector

Construction and Packaging Services of AAV

Large scale AAV production

Adeno-Associated Virus (AAV)
Adeno-associated virus (AAV) was discovered in 1965, as a contaminant of adenovirus (Ad) preparations, hence the name. It is replication-defective, non-enveloped small viruses (20nm) with a genome of single stranded DNA, from the parvovirus family. As 80-90% of humans are sero-positive with AAV2, most people treated with AAVs are non-pathogenic. Recombinant AAVs can infect both dividing and non-dividing cells and persist in an extrachromosomal state without integrating into the genome of the host cell.

The advantages of AAV compared to other viral vectors
● Long-term gene expression. rAAV genomes do not integrate into the host cellular genome, it persist in the nucleus in episomal forms. As cells replicate and divide, these episome will be lost. However, It can be expressed continuously for more than 6 months in tissues where cell division is not vigorous.
● Strong diffusivity. Due to small size and higher titer, rAAV has much higher diffusivity than adenovirus and lentivirus. And AAV can cross the blood-brain barrier, it is an ideal tool for neuron and glial cell infection.
● Specific expression can be achieved. AAV has a wide variety of promoters and serotypes, This enables AAV to recognize and infect different organs and cells, which make AAV as the first choice for animal experiments.
● High safety. AAV has not been found to cause disease to humans, and it is the safest viral vector approved by the FDA in US that can be directly used in human for gene therapy.
● low immunogenicity: When AAV is used to infect muscle, brain, eye and many others with local high dose, it is not easy to cause immune response;
● High stability. rAAV virus can be stored for 1 week at 4°C, and it is resistant to some reagents such as chloroform.
Viral vector AAV LV Ad
Genome ssDNA ssRNA (+) dsDNA
Coat Naked Enveloped Naked
Type Non-integrating Integrating Non-integrating
Infection Dividing and non-dividing cells Dividing and non-dividing cells Dividing and non-dividing cells
4.9kb 6kb 7.5kb
Potentially long-lasting Long-lasting Transient
Very Low Low High
Titer Up to 1012-13v.g/ml Up to 109TU/ml Up to 1012pfu/ml
Expression abundance High-level expression Moderate to high level expression High-level expression
Safety No pathogenicity has been found yet,
Has been approved by the EU and FDA,
Used as a carrier for gene therapy drugs
No pathogenicity has been found yet,
It has been used in CAR-T therapy in the human
May cause some coughing and runny nose

Packaging service
Process of AAV packaging

AAV virus packaging.(Melissa A. Kotterman.Nature Reviews et al.2014.)
Step1: Clone the foreign gene into a suitable viral vector.
Step2: The recombinant expression plasmid is co-transfected into the AAV-293 cells with pHelper (carrying adenovirus derived genes) and pAAV-RC (carrying AAV replication and capsid genes), which supply the trans-acting factors required for AAV replication and packaging in the AAV-293 cells. Recombinant AAV is assembled in packaging cells 2 to 3 days after transfection.
Step3: Recombinant AAV viral particles are prepared from infected AAV-293 cells and may then be used to infect a variety of mammalian cells. Generally, AAV viral particles are enriched in packaging cells, so collecting cells and then lysing them to release AAV particles into the supernatant can recover most of the AAV viral particles.
Step4: Concentrate and purify the virus-​containing supernatant above. The original supernatant contains many intracellular protein molecules and fragments. Purified virus is required for animal experiments, otherwise the required dose will not be achieved and side effects will be caused.
Step5: Quantitative PCR (qPCR) is one of the common methods to quantify the AAV vector titer. This method can obtain the physical titer of the AAV genome packaged in the viral particles. The infection titer of AAV varies greatly due to the infected cells, AAV coat protein and test conditions, and the experimental data in vitro cannot reflect the infection in vivo. Therefore, the physical titer obtained by qPCR is a more objective value.

How do I obtain virus packaging services?
BrainVTA provide professional,convenient and R & D products experienced service of AAV, ranging from small-scale to the large-scale, which help scientist accelerate the research process in circuit tracing, mechanisms of disease and treatment of disease.
So far, we have nearly 1000 pre-made AAV products with different serotypes and promoters. Meanwhile, we have offered 2000 customized AAV services for our clients with the following serotypes: AAV-1, AAV-2, AAV-5, AAV-6, AAV-8, AAV-DJ, AAV-PHP.eB, AAV-retro, AAV-anc80 and AAV-9. Artificially designed serotypes and vectors also can be produced on a case-by-case basis.
Please click Pre-Made AAVs for the in stock AAVs.
By clicking here for Custom-Made AAVs service.
Virus serotypes
BrainVTA currently offers the following serotypes: 1, 2, 5, 6, 8, 9, rh10, DJ, DJ8, PHP.B, PHP.eB, PHP.S, retro, pan, anc80. Novel peptide modified serotypes can also be produced on a case-by-case basis. Tissue specificity is determined by the serotype. If you Don't know which AAV serotype to use, see the following table to explore available AAV serotype to meet the efficient delivery of gene into the cells or tissues of interest.
 Serotype Tropism
AAV1 Muscle, heart, CNS, RPE, lung, skeletal muscle 
AAV2 In vitro, CNS, RPE, muscle, liver,CNS
AAV5 Lung, RPE, CNS, pancreas, adipose, liver
AAV6 Muscle, lung, heart
AAV8 Liver, muscle, RPE, CNS, adipose, pancreas
AAV-pan  Pancreas
AAV9 Lung, liver, muscle, heart, CNS, adipose, BBB
AAV.rh10 CNS, BBB, pleura
AAV-Rec2 Adipose
AAV.DJ In vitro, liver, heart, kidney
AAV.PHP.S DRG, heart, colon
● If you are not sure which serotype to choose, you can try AAV9 .
● If you use AAV for the first time, you can use the Rainbow Colors AAV for pre-experiment. By which you can compare the infection effects of different serotypes on the target tissue, and explore the best injection method, injection site, virus dosage, etc., thereby getting more ideal experimental results.

Rainbow Colors AAV
Rainbow Colors AAV is a set product that combines common serotypes suitable for different organizations.
Rainbow Colors AAV Serotype Volume
Eyes/other organs 1/2/5/6/8/9/DJ 10 μL/serotype
Nervous system 1/9/Retro/PHP. eB 10 μL/serotype
Liver/Kidney 2/8/9/DJ 100 μL/serotype
Heart/Vascular/Muscle 1/6/8/9 100 μL/serotype
*AAV1 is used for anterograde tracer.
AAV9 is used as anterograde tracer.
AAVRetro is used for retrograde tracer.
AAV PHP.eB is suitable for crossing the blood-brain barrier.

Tissue-specific promoters
BrainVTA offers various tissue-specific promoters. Listed below are the most commonly chosen promoters. Don' see the promoter of interest, Please inquire.
NO. Name Characteristics Sizebp
Strong promoters
1 CMV Ubiquitous 584
2 CAG Ubiquitous 1678
3 CBA( Cbh ) Ubiquitous 793
4 n Ef1α Short  Ef1α,Ubiquitous 493
5 Ef1α Ubiquitous 1179
6 hUbC Ubiquitous 1130
Inducible promoters
1 TRE3G Tetracycline(Tet)-inducible 350
Activity–dependent promoters
1 C-fos Immediate-early gene promoter 662
2 E-SARE Enhanced synaptic activity–responsive  element 954
Neuron-specific promoters
1 Mecp2 Truncated Mcep2 neuron specific 230
2 hSyn Mature neuron specific 485
3 CaMKIIa Specific expression inexci-tatory neurons in the neocortex and hippocampus 1293
4 PV GABAergic neuron subtypes 2396
5 SST GABAergic neuron subtypes 2597
6 VGAT GABAergic neuron 1800
7 mDlX GABAergic neuron 530
8 TH Dopaminergic neuron specific 299
9 TPH2 Tryptophan hydroxylase promoter 2042
10 ChAT Cholinergic neuron specific 1500
11 GFAP Astrocyte specific 2207
12 GFAP104 Short  GFAP 845
13 Cx30GJB6) Astrocyte specific 1505
14 CX3CR1 Microglia specific 1263
15 Mash1/Ascl1 Neural stem cell specific 1469
16 Nestin Neural stem cell specific 1394
17 L7/Pcp2 Purkinje cell 990
18 MBP Myelin basic protein promoter, efficient transduction of oligodendrocytes 1300
19 TRPV1 Transient receptor potential cation channel, subfamily V, member 1 2073
20 TRPV2 Transient receptor potential cation channel, subfamily V, member 2 1700
21 D1 D1 dopamine receptor 827
22 D2 D2 dopamine receptor 1211
23 OT Oxytocin promoter 2612
24 CRH Corticotropin-Releasing Hormone (CRH) promoter 456
25 PTH Human parathyroid hormone promoter 925
Eye-specific promoters
1 hGRK1                 Human rhodopsin kinase promoter 292
2 CAR         Cone arrestin gene promoter 523
3 Grm6                Bipolar cell specific expression 200
4 mRHOP        Rhodopsin gene promoter 524
5 ROH          Rhodopsin gene promoter 2200
6 Nrl                  Neural retina leucine zipper gene promoter 3200
7 RK         Rhodopsin kinase promoter 295
Liver-specific promoters
1 ALB Albumin enhancer promoter 1001
2 TBG Serine (or cysteine) peptidase inhibitor, clade A 410

Selection of vectors
BrainVTA provides a variety of AAV vectors, which can manipulate coding and non-coding genes through overexpression, RNAi and other technologies, such as lncRNA, microRNA, and circRNA. For more details, please contact [email protected].

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