Figure 2

Characterization of TgDHCR7 transgene expression. Panel A shows the tissue distribution of TgDHCR7 transgene mRNA as determined by RT-PCR. Amplification was performed with primers bounding the intron in the transgene. Note that expression was readily detectable on the liver tissues only. Panel B shows the profile of TgDHCR7 transgene expression during embryonic liver development, determined by RT-PCR. The samples were taken from embryonic days as indicated. Robust expression was detectable in liver tissue as early as E11.5. Panel C shows quantitation of transgene mRNA expression (TgDHCR7) relative to endogenous Dhcr7 (endo-Dhcr7) in liver samples by real-time RT-PCR. The transgene message was almost 5-fold greater than endogenous Dhcr7 message. The mean ± SD of Ct values of Tg-DHCR7 and endogenous Dhcr7 are shown in chart. Panel D shows protein expression of transgenic HA-tagged DHCR7 from the multiple tissues of TgDHCR7 (Tg) positive (+) and Tg negative (-) control mice. Membrane and cytosolic fractions from the multiple tissues of TgDHCR7-2 and TgDHCR7-3 positive animals (liver 1 and 2 respectively, lanes 3 and 4) and the livers of Tg negative control mice (liver 3 and 4, lanes 5 and 6) were prepared for immunoblotting analyses as described in Methods. The upper panel shows western blotting data using monoclonal anti-HA antibody (arrow denotes DHCR7 ~43 kDa). The asterisk at 48 KDa indicates immunoreactivity to IgG heavy chain in the samples. Middle and lower panels are immunoblots for calnexin (an ER membrane marker) and caveolin-1 (cav1, a plasma membrane marker), respectively, as controls. Panel E shows immunohistochemical staining with anti-HA antibody (left panel) and phase-contrast (middle panel) images of transgenic liver sections. No signal was detected with the antibody in control liver (right panel). T indicates liver portal triad.