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AdenoExpress™ PowerPoint PPT Presentation


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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™

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Adenoexpress l.jpg

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


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    AdenoExpress™

    Available with Biological Response Genes

    • Cytokines

      • GM-CSF

      • IFN-a

      • IFN-g

      • IL-2

      • IL-12


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    AdenoExpress™

    • Angiostatic genes

      • Angiostatin

      • Endostatin

  • Tumor suppressors

    • P53

  • Co-Stimulatory

    • B7.1


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    AdenoExpress™

    Two Formats Available


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    AdenoExpress™

    Coronal Plane Section of Rat Striatums

    Injected with Ad5.CMV-GFP

    Courtesy of Dr. Michael Y. Chen, NIH


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    AdenoExpress™

    Rat Spinal Cord

    Injected with Ad5.CMV-LacZ

    Courtesy of Dr. Michael J. Iadarola, NIH


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    AdenoExpress™

    Mature Dentritic Cells

    Infected with Ad5.CMV-GFP

    Courtesy of Dr. Peter Hevezi, EOS Biotech


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


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    Ad5.CMV5-GFP

    External and internal images of Ad5.CMV-GFP


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    Ad5.CMV5-GFP


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    Ad5.CMV5-GFP

    External whole-body image of Ad5.CMV5-GFP

    in the brain 24h after gene delivery.


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    Ad5.CMV5-GFP

    External whole-body image of Ad5.CMV5-GFP

    gene expression in the liver


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    Cumate Inducible Expression System


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    Cumate Inducible Promoter Regulatory Elements


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    Cumate Inducible Promoter Strategy 2


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    Applications of the Cumate Inducible Promoter


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    Generation of Recombinant Adenoviruses Expressing Toxic Proteins


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    Generation of Recombinant Adenoviruses Expressing Toxic Proteins


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    Inducible Expression in Stable Cell Lines


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    Adenovirus-Based Expression Libraries


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    Adenovirus-Based Expression Libraries


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    Adenovirus Custom Services


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    Adenovirus Custom Services

    From cloning of your cDNA into an Adenovirus transfer vector

    to

    cGMP Manufacturing


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    Adenovirus Custom Services

    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.


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    Adenovirus Custom Services

    Construction of a Recombinant Adenovirus

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

    • Plaque purification

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


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    Adenovirus Custom Services

    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.


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    Adenovirus Custom Services

    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.


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    Adenovirus Custom Services

    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.


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    Adenovirus Custom Services

    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.


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    Adenovirus Custom Services

    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.


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    Adenovirus Custom Services

    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.


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    Adenovirus Custom Services

    Recombinant Adenovirus Titration

    • Plaque Assay (Plaque Forming Units, PFU)

    • Tissue Culture Infectious Dose 50 (TCID50)

    • OD260 (Viral Particle, VP)


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    Adenovirus Custom Services

    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.


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    Adenovirus Custom Services

    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.


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    Adenovirus Custom Services

    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.


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    CONTRÔLE DE LA QUALITÉ


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    Plan

    • 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


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    Qu’est-ce que la qualité

    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


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    Qu’est-ce que le contrôle de la qualité

    • Vérification de la conformité du produit aux exigences spécifiées


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    Pourquoi le contrôle de la qualité?

    Assurer une reproductibilité du produit


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    Outils du contrôle de la qualité

    • Mode opératoire normalisé

    • Fiches analytiques

    • Assistant QC


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    Tâches de l’assistant QC


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    Qualités de l’assistant QC

    • Autonome

    • Structuré

    • Polyvalent

    • Minutieux

    • Appliqué

    • Honnête


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    Documentation et Assurance de la qualité

    • 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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