Generation of transgenic mice that overexpress human ubiquilin-1.

<p>(A) Schematic of the Thy1.2 expression construct used to generate ubiquilin-1 transgenic mice. Human ubiquilin-1 with an N-terminal FLAG tag was cloned in the appropriate orientation between the XhoI site of the Thy1.2 expression cassette. (B) Southern Blot of the first generation offspring of two founder mice (48 and 62). (C) Validation of a PCR genotyping protocol. Amplification of the transgene was only observed in mice that Southern blotting revealed to be positive. (D) Immunoblots of brain cortical lysates with an anti-FLAG antibody and for tubulin indicated that line 62 offspring express higher levels of FLAG-ubiquilin-1 than line 48. (E) Immunoblots of equal amounts of total brain lysates from 12 month-old WT mouse, 12 month-old Ubqln-1 48 transgenic mouse, 12 month-old Ubqln-1 62 transgenic mouse and end stage 15 week-old R6/2 transgenic mouse. The top panel was probed with a monoclonal anti-ubiquilin antibody (Invitrogen antibody clone 3D5E2) and the lower panel with a different monoclonal anti-ubiquilin antibody (Novus antibody clone 5F5). Note two immunoreactive ubiquilin bands are seen at ∼70 kDa and at ∼90 kDa, which we presume is a modified form of ubiquilin. Both blots were also probed for actin to ensure equal loading. (F) Cryostat sections of a Ubqln-1 62 transgenic mouse brain (a–f) and WT mouse brain (g-i) showing anti-FLAG antibody staining (Alexa 594, left panels) and corresponding DAPI staining (center panels) and the result of merging the fluorescent and DAPI signals (right hand panels). The brain sections shown are of the hippocampus (a–c and g–i) and cerebellum (d–f). Identical exposure settings were used for the left hand panels.