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.
Sargin et al. BMC Biology 2011 9:27 doi:10.1186/1741-7007-9-27