Tamoxifen-inducible ET2 expression and its effects on gene expression. (A) Diagram for induction of ET2 expression in vivo. In ROSA26-ET2 knockin mice, a construct was inserted downstream of the promoter of the ROSA26 gene. The construct contains a selection marker (pGK-NeoPGK-pA) and a transcriptional stop sequence (shown as a stop sign) flanked by 2 loxP sites (triangles), which are followed by the ET2 sequence plus the GFP sequence whose translation is mediated by an internal ribosome binding site (IRES). ROSA26-ET2 mice were crossed with ROSA26-CreERT2 mice, which produce a tamoxifen-inducible Cre. The double knockin mice were then treated with 5 doses of tamoxifen at 3 mg/mouse over 4 days to excise the selection marker and stop sequence, allowing expression of both ET2 and GFP. (B) Bone marrow cells from treated double knockin mice were harvested and lineage-depleted cells were obtained. Lineage-negative cells were sorted based on expression of GFP along with c-kit and Sca-1 markers. GFP-negative and -positive LSK and MP were collected based on the gates illustrated on the right. (C) Effect of ET2 on gene expression. A diagram shows the structure of ET2 protein and its proposed mechanism of action, which involves competition with Id proteins to dimerize with endogenous E proteins and subsequent binding to E boxes facilitating target gene transcription. To assess the function of ET2, real-time PCR assays were performed using total RNA isolated from cell populations sorted as described in panel B. Average levels of expression of indicated genes relative to that of β-actin in each indicated cell population are shown in bar graphs with SD. GFP-positive and -negative cells are considered to represent cells with or without ET2 expression.