• Adeno-Assoicated Virus (AAV) Packaging Service


    Vigene Biosciences has developed the most robust and professional AAV packaging service, ranging from crude small scale perfect for cell culture to large scale purified. AAV viral particles can be directly applied onto cells or in vivo work (animal injections).

    AAV packaging options

    AAV delivery of Cas9 and sgRNA

    Choice of serotype

    Choice of tissue specific promoter

    Ordering information

    Handling and storage of your AAV

    Additional Viral Resources

    AAV packaging options

    In addition to packaging customer provided plasmids, we offer full subcloning and plasmid prep services prior to viral particle production.

    Packaging Option

    Description

    Price

    Small Scale Crude Packaging

    500µl of rAAV at 1x10E12 GC/ml
    Coupled with Vigene broad selection of AAV vector cloning services, we provide crude AAV packaging service. The crude AAV virus is good for in vitro cell based experiments. Please send us 300-1000µg plasmid in TE buffer. For R/C send >150µg; for Helper >250µg. Please also see section on what to provide us.

    $545

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    Large Scale Packaging, in vivo ready

    500µl of rAAV at 1x10E13 GC/ml
    AAV virus produced from 109 Hek293 cells are purified by IDX gradient ultracentrifugation and resulted virus are concentrated to 500µl with virus titer no less than 1013vp/ml. The purified virus is good for in vivo animal research. Please send us 500-1000µg plasmid in TE buffer. For R/C send >150µg; for Helper >250µg. Please also see section on what to provide us.
    See section on working with your Virus for information on in vivo animal injections.

    $1445

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    Crude versus purified

    The purity of AAV particles is important for achieving stable and effective transduction into individual animals, as well as cultured cells. When introducing genes into animals (perfomed by in vivo transduction), it is important and necessary to use highly-purified AAV particles that do not contain impurities from the culture medium or any residual virus-producing cells. Furthermore, when transducing cultured cells, purified AAV particles eliminate the potential for negative effects of impurities.

    AAV packaging service inclusions

    ▪ Transfection

    ▪ Packaging: Small or Large-scale

    ▪ Purification (large scale only): performed using IDX gradient ultracentrifugation. We do not routinely use CsCl purification - if interested please contact us to discuss.

    ▪ Quality control assays: physical titer (vg/ml) as determined by QPCR and silver stain to assess virus titer and purity

    ▪ Vehicle: AAV particles are provided in F68/PBS

    Preparation of AAV particles to deliver Cas9 and sgRNA

    Genome editing using CRISPR/Cas9 is a powerful technique allowing creation of various gene knock-ins, knock-outs, and mutations in any cell, in a highly targeted manner, without foreign DNA introduction. The CRISPR/Cas9 technique, compared to previous forms of gene editing methods, such as TALENs and zinc finger nucleases, is simpler to implement as well as it offers higher efficiency bi-allelic gene modification.

    AAV delivery of CRISPR/Cas9 and sgRNA allows for genome editing in hard-to-transfect mammalian cells, including proliferating and non-proliferating cells. By choosing AAV delivery you reduce any off-target effects by precluding genomic integration and persistent Cas9 expression.

     

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    AAV ordering information

    What to provide Vigene for AAV packaging: Check list

    Large scale AAV packaging: minimum 500µg - 1000µg (preferably >1µg/ul) of your rAAV plasmid.

    Small scale AAV packaging: minimim 300µg (preferably >1µg/ul) of your rAAV plasmid.

    Clearly lable the tube(s) with plasmid name and DNA concentration(µg/µl).

    DNA should be purified using an endotoxin-free protocol.

    Plasmid DNA should be checked for purity: A260/280 of >1.8.

    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.

    Information about your gene of interest/insert along with any special requirements for handling e.g. toxicity, oncogenic, pro-apoptotic, enzymes used to release the insert, expected insert size etc.

     

    How do I send you my plasmid(s)?

    Mailing address

    Please address your plasmid(s) as follows:

    Vigene Biosciences
    Ref # [quote number]
    9430 Key West Ave
    Suite 105
    Rockville, MD 20850 USA

    Carrier options

    It is recommended to use an overnight express carrier (e.g. FedEx, UPS, DHL) instead of the postal service. Shipping from outside the USA: we also recommend overnight or another fast service using a carrier as this helps to minimize transit time and delays in your project.

    All this information, especially sequencing information (if needed for cloning/subcloning projects), should be emailed to orders@vigenebio.com.

    How much plasmid do I send?

    500-1000µg.

    For AAV packaging, we request that you send us 500-1000µg. We have received smaller quantities but we strongly recommend that you check with us before sending what you have. If you only have a very small ammount (1-5µg) then we can prepare a larger quantity of endotoxin-free DNA for you (please contact us for pricing).

    Is room temperature OK?

    Yes, if in TE buffer.

    Provided your plasmid is in TE buffer, sending plasmids at room temperature is perfectly acceptable. Please check with us about shipping options if you use other buffers, usually we will recommend using shipping in dry ice (or similar packaging) to reduce chance of DNA degradation.

    No plasmid? No worries.

    Vigene provides a full service from designing, cloning, to packaging and generating transduction-ready recombinant AAV particles. Service includes preparation, extraction, purification and titration.

    We provide the most customizable service; our scientists will work with you step-by-step and learn about your research goal to design and manufacture your ideal AAV particles.

     

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    How to select the appropriate AAV serotypes?

    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 (AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9). The following table lists most popular rAAV serotype and their tropism.

    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.

     

    Handling and Storage of Adeno-associated Virus

    It can be very useful to think of AAV as a rather large protein (MW= ~5,200,000). Given this MW, it is relatively stable but should be treated with at least as much care as any other proteins. AAV is also resistant to most proteases as well as a variety of chemical treatments that would normally inactive many other viruses. However, because of its large size, it has a large surface area which make it more likely to stick to hydrophobic surfaces and aggregate than smaller proteins.

    Both AAV and lentivirus are sensitive to freeze-thawing, where the titer drops with each subsequent freeze cycle. Some researchers have reported that they never freeze AAV, rather store at 4C with reported virus stablility years Manfreddson Lab, Michigan State. Here, at Vigene we highly recommend that researchers aliquot further and freeze at -80C for long term storage if the virus cannot be used within 1-2 weeks. AAV appears to retain its infectious activity for more than 10 years when stored at -80C.

    Some things to avoid with AAVs:

    ● Avoid exposure to environmental extremes (pH, chelating agents like EDTA, temperature, organic solvents, protein denaturants, strong detergents, etc.)

    ● Avoid introducing air into the sample by vortexing, blowing bubbles and similar operations (may result in protein denaturation).

    ● Avoid freeze and thaw multiple times. Although, AAV is more stable than many other viruses and can be frozen and thawed several times with minimal activity loss, it is best to avoid this practice.

    ● Avoid expose to “regular” plastics (especially polystyrene or very hydrophobic plastics) for prolonged periods in liquid phase. Most AAVs are very sticky and loss can occur if exposed to regular plastics, including tubes, cell culture plates, pipette tips, if not frozen. It is best practice to store thawed AAV in siliconized or low protein binding tubes. Pipetting should be perfomed with similar pipette tips. Pluronic F-68 used at 0.01%-0.1% in the formulation buffer will minimize sticking if regular plastics are used.

    ● Avoid diluting into low salt. Some AAVs, such as AAV2, aggregate in low salt and if the aggregates are large they will be non-infectious.

    Additional Viral Resources

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

     

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