Les souris sont identifiées par ablation d'im doigt lorsqu'elles sont âgées d'une semaine à dix jours. A 4-5 semaines d'âge, un morceau de queue est prélevé et digéré durant une nuit à 55°C dans une solution (Tris 100 mM pH 8,5 ; EDTA 5 mM ; SDS 0,2% ; NaCl 200mM) contenant de la protéinase K düuée à 100 pg/ml. Le lendemain, l'ADN génomique est extrait grâce au kit Puregene Core de Qiagen et 10 pg sont clivés avec différentes enzymes de restriction (EcoRI, BamHI, HindHI et Kpnl). L'ADN digéré est ensuite séparé par électrophorèse sur gel d'agarose 1%. Après la migration, le gel est traité comme suit : dépurination à l'HCl 0,5 N ; dénaturation dans im tampon de NaOH 0,5 M et NaCl 1,5 M ; neutralisation dans xme solution de Tris 0,5 M et NaCl 1,5 M ; et enfin lavage avec du SSC concentré 20x (NaCl 3 M; Citrate de sodium 0,3 M). L'ADN est alors transféré par capillarité sur une membrane de nylon (Flybond N+, GE Healthcare) à laquelle il se lie de manière covalente grâce à ime exposition de 3 minutes aux ultra-violets. La membrane est pré-hybridée dans ime solution de saturation (Denhart lOx ; SSC 4,5x ; SDS 2%) contenant 700 pl d'ADN de sperme de hareng dénaturé. Après minimum 1 heure à 65°C, la solution est remplacée par rme même solution mais contenant la sonde radioactive marquée au [a-^^P] dCTP, dénaturée. Cette sonde correspond aux 376 dernières paires de bases du promoteur CAG - elle est localisée dans l'intron. L'hybridation se déroule durant une nuit à 65°C puis la membrane est lavée avec une solution de SDS 0,1% et SSC 4,5x. Les résultats sont finalement analysés par autoradiographie (film XAR, Kodak). La carte de restriction pour les différentes constructions est fournie ci- après, Figure 33.
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