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AdenoExpress™. Available with Reporter genes: Ad5.CMV-GFP Ad5.CMV5-GFP Ad5.CMV-LacZ Null Vectors: Ad5.Null Ad5.CMV-Null Wild Type Viruses: Adenovirus Wild Type Serotype 5 Adenovirus Wild Type Serotype 2. AdenoExpress™. Available with Biological Response Genes Cytokines GM-CSF

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Adenoexpress
AdenoExpress™

Available with

  • Reporter genes:

    • Ad5.CMV-GFP

    • Ad5.CMV5-GFP

    • Ad5.CMV-LacZ

  • Null Vectors:

    • Ad5.Null

    • Ad5.CMV-Null

  • Wild Type Viruses:

    • Adenovirus Wild Type Serotype 5

    • Adenovirus Wild Type Serotype 2


  • Adenoexpress1
    AdenoExpress™

    Available with Biological Response Genes

    • Cytokines

      • GM-CSF

      • IFN-a

      • IFN-g

      • IL-2

      • IL-12


    Adenoexpress2
    AdenoExpress™

    • Angiostatic genes

      • Angiostatin

      • Endostatin

  • Tumor suppressors

    • P53

  • Co-Stimulatory

    • B7.1


  • Adenoexpress3
    AdenoExpress™

    Two Formats Available


    Adenoexpress4
    AdenoExpress™

    Coronal Plane Section of Rat Striatums

    Injected with Ad5.CMV-GFP

    Courtesy of Dr. Michael Y. Chen, NIH


    Adenoexpress5
    AdenoExpress™

    Rat Spinal Cord

    Injected with Ad5.CMV-LacZ

    Courtesy of Dr. Michael J. Iadarola, NIH


    Adenoexpress6
    AdenoExpress™

    Mature Dentritic Cells

    Infected with Ad5.CMV-GFP

    Courtesy of Dr. Peter Hevezi, EOS Biotech


    Visualizing gene expression by whole-body fluorescence imagingYang et al. (2000) Proc. Natl. Acad. Sci. USA 97, 12278-12282AntiCancer Inc, San Diego, CA


    Ad5 cmv5 gfp
    Ad5.CMV5-GFP imaging

    External and internal images of Ad5.CMV-GFP


    Ad5 cmv5 gfp1
    Ad5.CMV5-GFP imaging


    Ad5 cmv5 gfp2
    Ad5.CMV5-GFP imaging

    External whole-body image of Ad5.CMV5-GFP

    in the brain 24h after gene delivery.


    Ad5 cmv5 gfp3
    Ad5.CMV5-GFP imaging

    External whole-body image of Ad5.CMV5-GFP

    gene expression in the liver




    Cumate inducible promoter strategy 2
    Cumate Inducible Promoter imagingStrategy 2









    Adenovirus custom services1
    Adenovirus Custom Services Proteins

    From cloning of your cDNA into an Adenovirus transfer vector

    to

    cGMP Manufacturing


    Adenovirus custom services2
    Adenovirus Custom Services Proteins

    Cloning of Gene into an Adenovirus Transfer Vector

    • Subcloning of your gene of interest into an adenovirus transfer vector.

    • Verification of the orientation of the subcloned insert by restriction mapping.


    Adenovirus custom services3
    Adenovirus Custom Services Proteins

    Construction of a Recombinant Adenovirus

    • Production of a recombinant adenovirus by in vivo homologous recombination.

    • Plaque purification

    • Screening (Southern, Western, or by PCR).


    Adenovirus custom services4
    Adenovirus Custom Services Proteins

    Plaque Purification of a Recombinant Adenovirus Prior to Amplification

    • Plaque purification assay and isolation of a pure recombinant adenovirus (when Ad provided by customer).

    • Identification of a pure recombinant adenovirus by Southern, Western or by PCR screening.


    Adenovirus custom services5
    Adenovirus Custom Services Proteins

    Amplification of a Recombinant Adenovirus

    • Amplification of a recombinant adenovirus in 293 cells at a desired volume.

    • Infected cells are harvested and three freezing and thawing cycles are performed, followed by centrifugation to remove cell debris from recombinant adenovirus.


    Adenovirus custom services6
    Adenovirus Custom Services Proteins

    Double Cesium Chloride Gradient Purification of a Recombinant Adenovirus

    Amplified viral stocks should be purified in order to:

    • Remove all defective particles from the preparation,

    • Remove the condition media containing undesired contaminants as well as serum,

    • Resuspend the virus in a buffer more suitable for later use and long term storage,

    • Concentrate the virus to high levels.


    Adenovirus custom services7
    Adenovirus Custom Services Proteins

    Double Cesium Chloride Gradient Purification of a Recombinant Adenovirus

    • Purification on a CsCl step gradient followed by a continuous CsCl gradient.

    • Dialysis of viral stock against desired buffer and aliquoting.

      P.S. Purification of a virus is essential before undertaking any in vivo project.


    Adenovirus custom services8
    Adenovirus Custom Services Proteins

    Concentration of a Purified Recombinant Adenovirus

    • Concentration of purified virus via a filter centrifugation up to 1013 VP/mL.

    • Viral particle titration (VP) by OD260 measurement.


    Adenovirus custom services9
    Adenovirus Custom Services Proteins

    Identity Test of Viral Stock

    • Infection of non-permissive cells (ex. HeLa) with amplified viral stock.

    • Extraction of cellular proteins from infected cells.

    • Western blotting on extracted proteins.


    Adenovirus custom services10
    Adenovirus Custom Services Proteins

    Recombinant Adenovirus Titration

    • Plaque Assay (Plaque Forming Units, PFU)

    • Tissue Culture Infectious Dose 50 (TCID50)

    • OD260 (Viral Particle, VP)


    Adenovirus custom services11
    Adenovirus Custom Services Proteins

    Time Course and MOI Assay for Protein Production

    • Infection of HeLa cells using the recombinant adenovirus with three different MOIs.

    • Harvesting infected cells at two time points, typically at 48 and 72 hours.


    Adenovirus custom services12
    Adenovirus Custom Services Proteins

    Large-Scale Protein Production in HeLa Cells

    • Infection of HeLa cells in serum-free media with the recombinant adenovirus.

    • Protein expression in HeLa cells, according to characteristics of the recombinant adenovirus or custom specifications.

    • Harvesting infected cells and concentration by centrifugation.

    • Delivery of a cell pellet and/or culture supernatant.


    Adenovirus custom services13
    Adenovirus Custom Services Proteins

    Clinical Grade (cGMP) Production

    • Pilot project production of adenoviral vectors under GLP,

    • Adenoviral vector production, clinical (cGMP) grade,

    • Complete safety testing,

    • cGMP compliance and FDA registration.



    Plan Proteins

    • Qu’est-ce que la qualité

    • Qu’est-ce que le contrôle de la qualité

    • Pourquoi le contrôle de la qualité?

    • Outils du contrôle de la qualité

    • Tâches de l’assistant QC

    • Qualités de l’assistant QC

    • Documentation et Assurance de la qualité


    Qu’est-ce que la qualité Proteins

    Ensemble des propriétés et des caractéristiques d’un service ou d’un produit qui lui confèrent l’aptitude à satisfaire des besoins exprimés ou implicites


    Qu’est-ce que le contrôle de la qualité Proteins

    • Vérification de la conformité du produit aux exigences spécifiées


    Pourquoi le contrôle de la qualité? Proteins

    Assurer une reproductibilité du produit


    Outils du contrôle de la qualité Proteins

    • Mode opératoire normalisé

    • Fiches analytiques

    • Assistant QC



    Qualités de l’assistant QC Proteins

    • Autonome

    • Structuré

    • Polyvalent

    • Minutieux

    • Appliqué

    • Honnête


    Documentation et Assurance de la qualité Proteins

    • Vérification de la documentation pour s’assurer du respect des méthodes et des spécifications

    • Assurance de la qualité: ensemble des activités nécessaires pour donner une confiance adéquate que les produits satisferont aux exigences spécifiées


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