B. Cytométrie de flux
2. Expression de GaLV-FMG et fusions
L’expression de GaLV-FMG après infection ou transfection transitoire a été testée
dans les cellules 518.A2 et CHO.
Des vecteurs AAV-(GaLV-)FMG, de sérotypes 2 et 5 ont été utilisés pour transduire
les cellules 518.A2 et CHO, respectivement. Le choix des sérotypes avait été déterminé
auparavant avec le vecteur AAV-GFP (Cheong, thèse 2005). Les stocks de AAV-FMG ont
été produits par le « Vector Core » de l’hôpital universitaire de Nantes (titres et MOI
utilisées : voir tableau ci-dessous).
Un stock de AAV2-GFP (2.10
7u.i./ml), produit au laboratoire, a été utilisé comme
contrôle d’infection pour les deux lignées.
Les plasmides utilisés pour transfecter les cellules étaient pCR3.1eGFP et
pCR3.1GaLV, exprimant le transgène sous contrôle du promoteur CMV (0.5µg
d’ADN/2.5.10
4cellules au FuGENE).
L’infection ou la transfection a été réalisée 7h avant la fusion qui s’est déroulée sur la
nuit (+/-16h). Afin de déterminer l’expression de la protéine GaLV-FMG par les cellules
transfectées, infectées ou les CHO-FMG, elles ont été marquées avec un anticorps primaire
anti-FMG (anticorps monoclonaux générés par J-D Franssen, Euroscreen, Gosselies) et
colorées avec un anticorps secondaire anti-mouse-Ig-PE (Biosource) puis passées au
cytomètre de flux.
Particules totales/ml Unités infectieuses/ml MOI utilisée En particules totales (approx u.i.)
Infection des cellules
AAV2GaLV 3.7.10
9~3.7-7.4.10
71000 (~10-20) 518.A2
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