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AniRA Vectorologie

https://www.sfr-biosciences.fr/plateformes/cellular-and-dna-technologies/AniRA-Vectorologie/Vectorologie/leadImage

Purpose

The AniRA-Vectorology core facility provides technological expertise in retro and lentiviral vector production for the stable and effective transfer of nucleic sequences (gene, DNAc, Sh/miRNA) in cell lines, primary cells that are difficult to transfect (stem cells...) or in a whole organism. This system of transfer is used in many areas from basic to clinical research. Many applications are possible and the use of these vectors has been gradually extended to gene therapy, immunotherapy, vaccinology, transgenesis and virology.

AniRA-Vectorology is part of the regional AniRA core facility certified by the IBiSA label (IBiSA coordinates core facilities in the area of life sciences throughout France) and a member of the French Research Infrastructure Celphedia (a French National Research Infrastructure for animal models).

The AniRA-Vectorology core facility proposes is expertise and provides batches of characterized retrovirus and lentivirus vectors ready to use in C2 containment according to the biosafety requirements of French Haut Conseil des Biotechnologies. The productions are achived in C2 or C3 following in-house rules of the vectorology core facility. The main activity of the vectorology core facility is to produce MLV-, HIV-1 or SIV-based vectors.

 

More specifically, staff members of the AniRA-Vectorology provide assistance in:

  • Selection of vectors, for the cloning of the genes of interest and for assigning the vectors to the appropriate biosafety level.
  • Production of vectors for the overexpression of endogenous genes or for the ectopic expression of a gene in a cell line.
  • Production of vectors for gene knock-out (KO) encoding shRNAs against the gene of interest.
  • Production of vectors for conditional KO (inducible gene expression)
  • Production of C2 or C3 classified vectors.
  • Titer determination by FACS analysis (reporter protein), antibiotic selection or Q-PCR.
  • Lentiviral transgenesis, to generate transgenic mice (more information) Mettre le lien hypertexte comme dans le site en français
  • Training in production of vectors can be organized up on request.

 

More precisions about lentiviral transgenese

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Services

  • Production of retroviral vectors (MLV, HIV-1 and SIV) BSL2 or 3:

 

Production of lentiviral vectors is carried out by tri-transfection according to the method of calcium phosphate transfection, in an embryonic kidney 293T cell line. The three plasmids encode, respectively:

  • The envelope glycoprotein G of the vesicular stomatitis virus (VSV-G),
  • The viral structure and enzymatic proteins (gag and pol).
  • The viral vector encoding for the gene of interest (GFP, …).

 

  • Concentration of virions

 

  • Titration of infectious viral particles:

 

By transduction of HEK293T cells followed by:

  • Detection by flow cytometry (FACS) of the vector using a reporter gene coding a fluorochrome (GFP, mRFP, mKate...),
  • Selection with an antibiotic (puro, neo, zeo...) using a resistance gene present in the vector.
  • Quantification of integrated genome copy number by QPCR (check with the core facility for compatibility ).

We can also quantify physical particles by ELISA assays (capsid protein: p24, p27, p30).

 

For vector production, the applicant must complete an application form and sign the In-house rules of the Vectorology core facility: Charte d’utilisation des services de la Plateforme de Vectorologie

 

No application for production will be taken into consideration without:

  • - An Application Form (Formulaire de demande): completely filled, in Word format and sent by email.
  • - The vector with its detailed description, in electronic version for submission to the HCB (Haut Conseil des Biotechnologies) with the approved name of the gene identifiable in a database.
  • - GMO/ HCB Registration number.

 

 

Equipments

 

  1. - 2 Rotors SW41 et SW32 Beckman
  2. - 1 Centrifugeuse réfrigérée  Allegra 25R Beckman
  3. - 2 PSM type 2
  4. - 2 Lots de pipettes Eppendorf
  5. - 1 Incubateur (en P3)
  6. - 1 Lecteur de plaques ELISA
  7. - 1 Bloc chauffant
  8. - 1 Centrifugeuse à micro tubes réfrigérée Eppendorf

 

How to contact us/where to find us:

 

  1. Bat. LR5 - Equipe: EVIR
  2. Ecole Normale Supérieure de Lyon
  3. 9 rue du Vercors
  4. 69007 Lyon
  5.  

For further informations:

  1. Caroline Costa Fejoz: Facility manager
  2. Tel : 04 72 72 87 28
  3. Mail : caroline.costa@ens-lyon.fr
  4. Pour toute demande de production de vecteurs, merci de  télécharger le fichier de demande de productions, le compléter et renvoyer au contact suivant :plateau.vectorologie@ens-lyon.fr

 

Training

Production of retroviral vectors: theory, labwork and biosafety (can be organized on request).

 

Tarifs

  1. Tarifs accessibles en intranet (équipes contributrices et équipes académiques) or on request.

 

Documentation

  1. Fichier de demande de production de vecteur

 

Links

  1. Guide 'O.G.M. en milieu confiné'
  2. Guide Haut Conseil aux Biotechnologies
  3. Un guide des risques biologiques (CNRS)
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