Figure 1

Construction and characterization of HA-cEPOR TG mice ('TG1'). (A) Construct HA-cEPOR used for production of HA-cEPOR TG mice. HA-cEPOR, flanked by a hybrid intron at the 5' end and a polyadenylation signal at the 3' end was placed under the control of the α-CaMKII promoter. (B) Forebrain-specific expression of HA-cEPOR transgene revealed by immunohistochemistry. A monoclonal antibody against the HA-tag was used to stain coronal sections of the hippocampus. Expectedly, HA-cEPOR expression is absent in WT mice [(a) and (b) are magnifications of the respective regions of interest]. In TG mice, HA-cEPOR expression is restricted to pyramidal neurons of the cortex (c), CA1 (d) as well as CA3 subregions of the hippocampus and granular layer of the dentate gyrus. Scale bars; 100 μm and 500 μm. (C) Tissue-specific expression of HA-cEPOR mRNA (top) and protein (bottom) in TG mice. TG mRNA expression was detected by PCR using TG specific primers, yielding a 362 bp product. Western blot analysis of HA-cEPOR using a monoclonal antibody against HA-tag revealed a 64 kDa band. HA-cEPOR mRNA and protein were expressed in cortex (CX) and hippocampus (HP) of TG mice but not in cerebellum (CB) or in peripheral tissues (LIV: liver; KID: kidney). GAPDH was used as the internal control for both mRNA (431 bp) and protein (36 kDa) expression analysis. (D) Developmental regulation of the HA-cEPOR transgene. HA-cEPOR transgenic mRNA and protein expression was not seen in fetal tissue ('embryonic' day 12 and 17), but detected at early postnatal days (P0, P14) and remained constant until adulthood.