Figure 2

Construct design and expression of the RIP-RacN17 transgene. (A) Dominant negative Rac1 (RacN17) was inserted in a vector containing the Rat Insulin Promoter (RIP). A C-myc-tag (red) fused to the N-terminal end of RacN17 enabled monitoring of expression of the transgene. Rabbit β globin intron (βgi) and poly A (βgpA) was inserted to enhance the expression of the transgene. (B) An immunoblot on islets from adult mice demonstrating expression of endogenous Rac1 in wild-type (Wt) islets and expression of endogenous Rac1 and slightly larger RacN17 in transgenic (Tg) islets. (C) Western blot quantification of protein shows that RacN17 is expressed at approximately four fold higher levels than endogenous Rac1 in isolated adult islets (n = 5). (D-L) Immunofluorescence staining on frozen sections with antibodies against C-myc (red) and insulin (green) at E17.5 (D-F) and in the adult (G-I). The transgene is selectively expressed in β cells, and the expression levels tend to vary within the β cell population, especially in the adult. (J-L) Adult wild-type islets show no labelling with C-myc. Error bars represent standard deviation from the mean (± s.d.). Bars, 50 μm.