Generation and analysis of Hes1 transgenic mice.

<p>(A) Copy numbers of the Hes1 transgene determined using real-time PCR with primers as depicted by the arrows in the diagram of the transgenic construct. HPC4-tag was added to the N-terminus of Hes1. Numbers of Hes1 coding sequences in the H5 and H12 lines of Hes1 transgenic mice are shown as relatives to that of WT mice. (B) Hes1 protein expressed by total thymocytes from WT and the H5 and H12 lines of Hes1 transgenic mice were detected with antibodies against Hes1 or the HPC4 tag. Levels of tubulin were used as loading controls. (C) Comparison of total Hes1 mRNA levels encoded by the endogenous gene and transgene in thymocyte subsets sorted from WT and Hes1 (H12) transgenic mice. The level of Hes1 in each sample was normalized against that of β-actin. Expression levels relative to that in WT DP cells are shown in log scale. The fold of increase in Hes1 transgenic cells relative to their wild type counterparts is labeled on top of the bars. Data shown in (A) and (C) are means±SD of triplicates. (D) Electrophoretic mobility shift assay for E-box DNA-binding activity. Nuclear extracts were prepared from sorted DN thymocytes from the indicated mouse strains at the age of 3–4 weeks, and incubated at room temperature with ³²P–labled probes and 1 µg of poly(dI:dC) as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006678#pone.0006678-Kim2" target="_blank">[51]</a>. For supershift assays, 1 µl of anti-E47 antibodies was added at the end of binding reaction and incubated for 5 minutes. DNA binding and super-shifted complexes are indicated by closed and open arrows. Oct-1 binding was used as a control.