Wdr5 Knockdown in Pancreatic Islets

Three different pLKO vectors containing short hairpin constructs targeting Wdr5 under control of the hU6 promoter, and a scramble shRNA construct, were purchased from OpenBiosystems. These shWdr5 expressing vectors were prescreened for their ability to suppress Wdr5 in MIN6 cells. Subsequently, the optimal targeting sequence that suppressed Wdr5 by ~90%, and the scramble sequence, were inserted into pAdTrack using InFusion cloning (Clontech) and sequence verified. These were then used to make the pAdV-shWdr5 and pAdV-shScramble adenoviruses using the pAdEasy system as previously described [69 (link)]. Islets were transduced with these adenoviruses at the indicated MOI’s for three hours and 48 hours later were treated with or without IFNγ, Il-1β and TNFα, as indicated, for a subsequent three hours. GSK-J4 (Sigma-Aldrich) or DMSO was used to treat islets at the concentrations indicated with or without IFNγ, Il-1β and TNFα for three hours.