Retrovirus production and infection
The YAP cassette was cloned from pBabe-YAP1 (Addgene) into the retroviral vector pWzl. Short hairpin RNA interference sequences targeting IGF2 at the 3′ or 5′ end of the coding sequence were designed using the Cold Spring Harbor Laboratory RNAi OligoRetriever database and ligated into pSHAG vector, then transferred to MSCV retroviral vector and produced as described (Nahle et al 2008). A control scrambled shRNA retrovirus was also prepared. 293 EBNA (Invitrogen) packaging cells were co-transfected with gag-pol and VSVg packaging plasmids plus pWzl-eGFP, pWzl-YAP1, pMSCV-IGF2shRNA or a control scrambled shRNA using Fugene 6 transfection reagent (Roche). The media was changed 12 hours after transfection and supernatants (8 mL) were harvested at 24 and 48 hours and filtered through 0.45 μm syringe filters. For infection, Shh-treated CGNPs were exposed to the viral supernatants for 3 hours. Viral supernatant was then removed and replaced with fresh medium + Shh.