• AAV Viral Transduction System


    Vigene offers a wide range of rAAV Services and packaging service: ranging from small crude scale to the custom large purified scale. Vigene Biosciences has developed the most robust and professional AAV packaging service, ranging from small crude scale to the custom large purified scale. Provides high-quality and high-titer viral services. Unfamilair with how AAV works, see our section on introduction to AAV.

    Robust AAV production

    AAV cloning and packaging

    Multiple serotypes

    AAV delivery of Cas9 and sgRNA

    Tissue-specific promoters

    Custom projects: full subcloning and plasmid prep services

    Trans-splicing system for large gene delivery

    AAV and rAAV

    Adeno-associated virus (AAV) is a small, single-stranded DNA virus. AAV is thought to be non-pathogenic to humans, with replication possible in the presence of a helper virus, such as Adenovirus. They are not permanently integrated into the host genome. These features make AAV a useful tool for gene delivery into a wide variety of cell types and an attractive vector for gene therapy.

    Common use: gene delivery in-vivo because of their mild immunogencity

    The genome of engineered recombinant AAV (rAAV) vectors contain no pathogenic genes. Inverted terminal repeats (ITR) are present making it impossible for viral self-replication. This “gutless” rAAV has been FDA evaluated as a safe vector for use in human gene therapy.

    Please note: that Vigene does not offer wild type AAV viruses or their controls.

    rAAV services

    Adeno-Associated Virus (AAV) Cloning Service
       ▪ AAV Human cDNA ORF Cloning
       ▪ AAV shRNA Cloning
       ▪ FLEX-ON of Cre dependent inducible expression
       ▪ Trans-splicing system for large gene delivery

    Adeno-Associated Virus (AAV) Packaging Service
       ▪ Small Scale Crude AAV Packaging Service
       ▪ Large Scale custom AAV Packaging Service
       ▪ AAV delivery of Cas9 and sgRNA

    Premade rAAVs

    Control Viruses
       ▪ Fluorescent proteins: GFP, RFP
       ▪ Cre recombinase
       ▪ Tissue-specific promoters

    Mammalian Expression Vectors
       ▪ Shuttling Vectors
       ▪ shRNA Vectors

    AAV Biosensors and Optogenetic Tools New!
       ▪ View cellular activity in living tissue
       ▪ Ready-to-use AAV vectors

    ZIKA ORFs New!
       ▪ 14 ZKV ORFs as plasmid DNA, AAV, adenovirus
       ▪ Complete ORF Set or single gene

    Additional Viral Resources

    Ordering Instructions: Shipping and payment information
    Product Manuals: How to use your virus
    FAQs : Answers to commonly asked AAV questions
    Publications: showcasing Vigene products and services
    Recipient instructions: information on viral safety and storage

     

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    AAV Viral Production Overview

    Production

    AAV is produced in-house by our scientists. Transfections are conducted using pAV-FH AAV vector, the Ad Helper vector, and a vector encoding the rep and serotype-specific cap. All of our AAV preparations are purified by gradient ultracentrifugation. We concentrate to purity and provide titers that can be used in in vivo studies. Click on the image below to view at higher resolution.

    Titer determination

    The virus titer is determined by viral genome copy number in 1ml sample by real-time quantitative PCR and compared to a standard curve of a plasmid sample of known concentration. Titering is performed on AAV that has been stored at -80C then thawed. Primers targeting the ITR are used and amplicon detection is made with SYBR green. The following data shows the titering results using the AAV-GFP virus.

     

    aav_virus_titer

     

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    Quality Control

    Purity

    AAV protein components are VR1 82kDa, VR2 72kDa and VR3 62kDa. We analyze our viral preparations using polyacrylamide gel electrophoresis (PAGE) followed by silver staining. These methods allow us to determine the molecular weight and relative intensity; Good AAV purification should only show three major protein bands by PAGE. The following image shows the protein components in our purified AAV virus.

    Protein_components_of_purified_AAV_virus

    Selecting AAV serotypes

    Serotype determines tissue specificity and is classified by the type of capsid structural protein; Serotype 2 (AAV2) is the most studied. AAV2, themost popular serotype, offers a broad range of tissue infectivity for research purposes.

    So far there are 11 AAV serotypes described, they all have different tropism and can infect cells from multiple diverse tissue types. Tissue specificity is determined by the capsid serotype. The selection of the right serotypes is critical for the efficient delivery of gene into the cells or tissues of interest. Vigene offers AAV cloning and packaging in multiple serotypes.

    Table. Availabe AAV serotypes and their tropism

    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.

     

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