• Frequently Asked Questions



    A selection of commonly asked questions relating to Vigene products and services. If you cannot find the answer you are looking for please contact us. We are more than happy to assist.

     

    AAV Collection

    What AAV serotype do you provide and which serotype should I use for my experiment?

    We currently provide AAV serotype AAV1, AAV2, AAV5, AAV7, AAV6, AAV8, AAV9 & AAV-DJ; see table as suggested by literatures.

    Please also refer to the transfection efficiency vs different cell type table to better determine which serotype works better for you.

    What are adeno-associated virus (AAV), recombinant AAV(rAAV) & helper free rAAV?

    The adeno-associated virus (AAV) is a small, icosahedral and nonenveloped virus that belongs to Parvoviridae family. Helper virus, such as Adenovirus or Herpes virus, is usually required for a productive infection to occur. AAV does not encode its own polymerase so its replication process relies on host cell polymerase activities.

    AAV is not currently known to cause any disease and has very mild immune response. Being able to infect both dividing and non-dividing cells, it incorporates its genome into that of the host cells and only replicates in the presence of a helper virus, most commonly adenovirus or herpes simplex virus. These features make recombinant AAV a very attractive tool for gene delivery to a wide variety of cell types and useful vector for gene therapy.

    Recombinant AAV is the artificial AAV without any AAV rep and cap genes which encode viral replication and structural proteins, respectively. In rAAV, rep and cap are replaced with a gene or construct of interest flanked by the ITRs for replication and packaging.? Efficient packaging of rAAV can be performed with constructs ranging from 4.1 kb to 4.9 kb in size.

    Because AAV is a replication defective virus, its replication depends on other helper virus such as adenovirus and herps virus.

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

    Purified AAV vectors are highly stable at temperatures of 4C and lower. It is recommended that you aliquot your products upon receipt, storing at -80C. Once an aliquot is thawed it may be stored, short term without significant loss of biological activity at 4C, for less than 3 weeks .

    What is the Biosafety requirement for using AAV?

    Recombinant AAV constructs produced in the absence of a helper virus and encodes no tumorigenic gene 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.

    Is recombinant AAV safe?

    To date, AAV is not linked to any human disease. For wild type AAV, replication is at extremely low efficiency, without the presence of helper virus, such as adenovirus. The recombinant AAV (rAAV) composed by several plasmids (cis plasmid, Helper plasmid, rep/Cap plasmid). Cis plasmid and Helper do not share any regions of homology with the rep/cap-gene containing plasmid, the likelihood for a recombinant AAV to replicate is theoretically impossible.

    What is the cloning capacity for recombinant AAVs?

    AAV has a packaging capacity of ~4.7Kb. When the length of inserted DNA between the 2 ITRs is close to the maximal allowed, i.e., 4.7Kb, the packaging efficiency decreases significantly. For instance, for gene over-expression from cDNA, since the CMV-poly(A) signal element is about 1Kb, so the maximal allowable cDNA length is about 3.2kb, whereas if GFP co-expression (about 0.8kb) is considered, the allowable capacity is about 2.4kb.

    What are the advantages of gene delivery by rAAV?

    AAV has the capacity to produce high titer virus in dividing and non-dividing cells and potential for long-term gene transfer with minimum immnunogenicity.

     

    Table 1 Relative in vitro infectivity of different AAV serotypes

    Cell lineAAV1AAV2AAV5AAV6AAV8AAV9
    Huh-7 13 100 0.1 10 0.7 0
    HEK293251000.150.70.1
    HeLa31006.710.20.1
    HepG231001.750.3ND
    Hep1A201000.110.20
    911171000.1170.1ND
    CHO10010033350101
    COS3310051420.5
    MeWo101006.71010.2
    NIH3T3101000.3100.3ND
    A549141000.5100.50.1
    HT1180201000.3330.50.1
    Monocytes11111001251429NDND
    Immature DC25001002222857NDD
    Mature DC 2222 100 333 3333 ND ND

     

    Table 2 AAV serotypes and preferred/main target tissue

    Serotypes differ mainly in surface protein (capsid). Choosing the most suited serotype for your project enables more robust tissue specific gene expression, reducing immune response.

    AAV
    Serotype
    Tissue Tropism ( indicates recommended application)
    Muscle Liver Lung Brain Retina Pancreas Kidney
    AAV1 Neurons and glial cells
    AAV2
    AAV5 Lung alveolar cells Neurons and glial cells
    AAV6
    AAV7 Neurons
    AAV8 Neurons
    AAV9 Neurons
    AAV-DJ A mix of 8 naturally occurring serotyes.
    Efficient for various types of human tissues and organs.