Post on 03-Apr-2015
- 1 -
AdenoExpress™
Available withReporter 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
- 2 -
AdenoExpress™
Available with Biological Response Genes
CytokinesGM-CSF
IFN-
IFN-
IL-2
IL-12
- 3 -
AdenoExpress™
Angiostatic genesAngiostatin
Endostatin
Tumor suppressorsP53
Co-StimulatoryB7.1
- 4 -
AdenoExpress™
Format Concentration
(VP/mL)
Volume
(μL)
Total Qty
(VP)
Small 1X1011 100 1X1010
Large 1X1013 50 5X1011
Two Formats Available
- 5 -
AdenoExpress™
Coronal Plane Section of Rat Striatums Injected with Ad5.CMV-GFP
Courtesy of Dr. Michael Y. Chen, NIH
- 6 -
AdenoExpress™
Rat Spinal Cord Injected with Ad5.CMV-LacZ
Courtesy of Dr. Michael J. Iadarola, NIH
- 7 -
AdenoExpress™
Mature Dentritic CellsInfected with Ad5.CMV-GFP
Courtesy of Dr. Peter Hevezi, EOS Biotech
Visualizing gene expression by whole-body fluorescence imaging
Yang et al. (2000) Proc. Natl. Acad. Sci. USA 97, 12278-12282
AntiCancer Inc, San Diego, CA
- 9 -
Ad5.CMV5-GFP
External and internal images of Ad5.CMV-GFP
- 10 -
Ad5.CMV5-GFP
- 11 -
Ad5.CMV5-GFP
External whole-body image of Ad5.CMV5-GFPin the brain 24h after gene delivery.
- 12 -
Ad5.CMV5-GFP
External whole-body image of Ad5.CMV5-GFPgene expression in the liver
- 13 - Cumate Inducible Expression System
- 14 -
Cumate Inducible Promoter Regulatory Elements
- 15 -
Cumate Inducible Promoter Strategy 2
Applications of the Cumate Inducible
Promoter
- 17 -
Generation of Recombinant Adenoviruses Expressing Toxic
Proteins
- 18 -
Generation of Recombinant Adenoviruses Expressing Toxic
Proteins
- 19 -
Inducible Expression in Stable Cell Lines
- 20 -
Adenovirus-Based Expression Libraries
- 21 -
Adenovirus-Based Expression Libraries
- 22 -
Adenovirus Custom Services
- 23 -
Adenovirus Custom Services
From cloning of your cDNA into an Adenovirus transfer
vector
to
cGMP Manufacturing
- 24 -
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.
- 25 -
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).
- 26 -
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.
- 27 -
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.
- 28 -
Adenovirus Custom Services
Double Cesium Chloride Gradient Purification of a Recombinant Adenovirus
Amplified viral stocks should be purified in order to:
1. Remove all defective particles from the preparation,
2. Remove the condition media containing undesired contaminants as well as serum,
3. Resuspend the virus in a buffer more suitable for later use and long term storage,
4. Concentrate the virus to high levels.
- 29 -
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.
- 30 -
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.
- 31 -
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.
- 32 -
Adenovirus Custom Services
Recombinant Adenovirus Titration
1. Plaque Assay (Plaque Forming Units, PFU)
2. Tissue Culture Infectious Dose 50 (TCID50)
3. OD260 (Viral Particle, VP)
- 33 -
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.
- 34 -
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.
- 35 -
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.
- 36 -
- 37 - CONTRÔLE DE LA QUALITÉ
- 38 -
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
PlanPlan
- 39 -
Qu’est-ce que la qualitéQu’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
- 40 -
Qu’est-ce que le contrôle de la qualitéQu’est-ce que le contrôle de la qualité
Vérification de la conformité du produit aux exigences spécifiées
- 41 -
Pourquoi le contrôle de la qualité?Pourquoi le contrôle de la qualité?
Assurer une reproductibilité du produit
- 42 -
Outils du contrôle de la qualitéOutils du contrôle de la qualité
Mode opératoire normalisé
Fiches analytiques
Assistant QC
- 43 -
Tâches de l’assistant QCTâches de l’assistant QC
C on fo rm e
R ecom m en ce le tes t
H ors sp é c ifica tion s
V é rifica tion d es sp é c ifica tion s
L ec tu re d es ré su lta ts
E ffec tu e le tes t
H ora ire d es tes ts
R eço it u n é ch an tillon
- 44 -
Qualités de l’assistant QCQualités de l’assistant QC
Autonome
Structuré
Polyvalent
Minutieux
Appliqué
Honnête
- 45 -
Documentation et Assurance de la qualitéDocumentation 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