• 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.

    Parkinsons Disease AAV Viral Vectors

    Coming soon

    Vigene has proudly partnered with The Michael J. Fox Foundation (MJFF) to provide high-quality reagents to support Parkinson’s disease research. A selection of viral vectors will be made available to researchers for a small fee thanks to the generosity and involvement of MJFF. Questions regarding the reagent tools can made using our contact form.

    Robust AAV production

    AAV packaging and purification

    AAV custom cloning

    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

    Vigene's Quality Assurance

    Vigene's Quality Control

    Don't know which AAV serotype to use? Try GFP AAV serotype test kit to optimize your experiments. See AAV serotypes section to explore available AAV serotypes, tissue tropisms and which human primary cell lines suites which AAV serotypes.

    Don't know which promoter to use? View our Tissue-specific promoters table and explore your options. If you don't see the one you are after please contact us.

    AAV Services: Turnaround time, Titer and Price

    The turn around times indicated in the table do not include DNA amplification. To qualify for AAV packaging without DNA amplification, the following amounts of transfer vector containing the gene of interest must be supplied:

    ● for a titer of 109 GC/ml minimum 75µg;

    ● for a titer of 1012 GC/ml minimum 500-600µg;

    ● for a titer of 1013 GC/ml minimum 500-1000µg.

    Please note: It is good practice to check your AAV ITR plasmids for recombination by digestion with SmaI or XmaI. Each ITR contains two SmaI/XmaI sites. RE digestion will cut out your insert. If you notice excessive amounts of linearized full-length plasmid then recombination has occurred.

    Viral Service

    Expected Minimum Titer

    Price

    Turnaround time

    Viral Vector
    Construction/Cloning and/or DNA amplification

    Please inquire

    7-30 days

    AAV
    Packaging

    Small scale: 1x1012sup>GC/ml, 500µl
    Large scale: 1x1013sup>GC/ml, 500µl

    $545

    $1398

    7-14 days

    Adeno-Associated Virus (AAV) Cloning Service

    For AAV cloning and packaging we recommend using the pAV-FH - download pAV-FH sequence file here. The pAV-FH has Amp resistance; restriction Sites: AsiSI M1uI. View cloning scheme. Explore additional shuttling vectors.

     

    Cat.# Vector Selection Promoter Tag Tag Position Price

    P100034

    pAV-FH

    Ampicillin

    CMV

    Flag-His

    ORF

    $249


     

    For adenoviral cloning, Vigene Biosciences has pioneered the shuttling vector: pEnter Entry Vector. The pEnter entry vector is compatible with a variety of destinations vectors, including AAV, adenovirus, lentivirus, and shRNA vectors. You may also Download pENTER sequence. Explore adenoviral cloning and packaging.

    ● AAV Human cDNA ORF Cloning

    ● AAV shRNA Cloning

    ● FLEX-ON of Cre dependent inducible expression

    ● Trans-splicing system for large gene delivery

    Explore cloning options

     

    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 Products

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

    Mammalian Expression Vectors
       ▪ Shuttling Vectors
       ▪ shRNA Vectors

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

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

    AAV delivery of Cas9 and sgRNA

    CRISPR is the acronym for Clustered Regularly Interspaced Short Palindromic Repeats. CRISPR sequences were first identified in the genome of Escherichia coli (E. coli), and found to function as part of an RNA-based adaptive immune system, targeting and destroying genetic parasites at the DNA level.

    ● Viral delivery of Cas9 and a sgRNA allows for genome editing in hard-to-transfect mammalian cells, this includes proliferating and non-proliferating cells.

    ● AAV delivery of Cas9 precludes genomic integration as well as persistent expression of Cas9, thereby reducing any off-target effects.

    Recommended reading - Adeno-Associated Virus-Mediated Delivery of CRISPR-Cas Systems for Genome Engineering in Mammalian Cells, CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox.

    Vigene can assist with your CRISPR needs, simply detail your approach using our contact form.

    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 Assurance

    Vigene's quality assurance policy ensures that the initial titer is provided in GC/mL as determined by qRT-PCR. If the guarnateed titer is not obtained in the first instance, Vigene will repeat the packaging/purification at no cost to the researcher as a one time repeat. Additional charges will be applied if subsequent steps are needed to obtain the desired titer, these will be discussed with the researcher before proceeding with the project. Sometimes the gamble is too risky to proceed with provided plasmids and we will do our best to guide you in the best options for your research goals. Please note that additional charges will apply in the following situations:

    Constructs over packaging capacity. For ssAAV this is ~4.7 kb from ITR to ITR and for scAAV this is ~2.2 kb from ITR to ITR;

    Involve a novel or modified capsid;

    Constructs where the sequence does not match the ITR digest. We highly recommend that you perform your own ITR digest before submission of a transfer plasmid. This is especially important if you have obtained plasmids from another lab. This can easily be done by digestion with SmaI or XmaI. Each ITR contains two SmaI/XmaI sites. RE digestion will cut out your insert. If you notice excessive amounts of linearized full-length plasmid then recombination has occurred;

    Shuttle backbone size. When the shuttle backbone is very small compared to transgene cassette leading to inappropriate packaging of the shuttle backbone.

    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

    Tissue Tropism ( indicates recommended application)

    AAV Serotype

    Muscle

    Liver

    Lung

    Brain

    Retina

    Pancreas

    Kidney

    Heart

    AAV1

    Neurons and glial cells

    AAV2

    AAV3

    AAV4

    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.

     

    Table. Human Primary Cell Lines vs AAV Serotypes

    Adapted from Ellis et al 2013, Virology Journal. Cells were analyzed using flow cytometry at 48h post-infection. MOI: 100,000 vg/cell. Legend shows the percentage of GFP positive cells.
    Legend: -, 0-5%; +, 5-40%; ++, 40-80%; +++, >80%.

    Cell lines

    AAV1

    AAV2

    AAV3

    AAV4

    AAV5

    AAV6

    AAV7

    AAV8

    AAV9

    BJ Fibroblasts

    +

    +

    +

    -

    -

    +

    -

    -

    +

    BJ hTERT Fibroblasts

    ++

    +

    +

    -

    -

    +

    -

    -

    -

    ES Cell

    -

    +

    ++

    -

    -

    +

    -

    -

    -

    HUVEC

    +++

    +++

    ++

    -

    +

    +++

    +

    +

    +

    Keratinocyte

    ++

    +

    +

    -

    -

    +++

    -

    -

    -

    Hematopoietic Progenitor

    -

    -

    -

    -

    -

    +

    -

    -

    -

     

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    Tissue-specific promoters

    Cat.#Promoter
    Name
    SizeDescription
    PM10001 ALB 2.4kb Liver specific 10 timer stronger than CMV after 10 weeks
    PM10002 GFAP104 845bp Hybrid of EF1a and GFAP
    PM10003 CAG 944bp Strong promoter, ubiquitous expression in vivo
    PM10004 CamKIIa 1.2kb Specific expression in excitatory neurons in the neocortex and hippocampus
    PM10005 EF1A 1.2kb Ubiquitous, weaker than CMV but better for in vivo
    PM10006 CK1.3 1.1kb
    PM10007 CK0.4 217bp Calcium/Calmodulin-dependent kinase II alpha
    PM10008 GFAP 2.0kb Specific in astrocyte
    PM10009 MBP 1.3kb Myelin basic protein promoter, efficient transduction of oligodendrocytes by adeno-associated virus type 8 vectors
    PM10010 EFFS 253bp A short version EF1A
    PM10011 TBG 460bp Homo sapiens serpin peptidase inhibitor, clade A
    PM10012 aMHC 0.4kb Mouse myosin heavy chain alpha promoter
    PM10013 cTNT 702bp Specifically transduce cardiomyocytes
    PM10014 Synapsin 471bp Specific in neuron
    PM10015 Mecp2 230bp Truncated Mcep2 neuron specific
    PM10016 c-fos 1.7kb Activity-dependent promoter
    PM10017 MCK 1.35kb Muscle creatine kinase promoter/enhancer
    PM10018 UBC 1.1kb Ubiquitous, weaker than CMV but better for in vivo
    PM10019 PGK 400bp Ubiquitous, weaker than CMV but better for in vivo
    PM10020 Somatostat 1.2kb Restricting expression to GABAergic neuron
    PM10021 Rpe65 700bp Retinal Pigment epithelium-specific expression in vivo and in vitro
    PM10022 Insulin1 1.0kb Specific in beta- cells of the pancreas
    PM10023 3Xenhancer McK 728bp Much stronger than CMV in muscle, inactive in nonmuscle cell lines and mouse liver
    PM10024 NSE 1.3kb Neuron-specific enolase promoter

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