Establishment of <i>G1HRD-idEpoRic</i> Transgenic Mouse Lines.

<p>(A) Schematic structure of the transgene product, idEpoRic (inducible dimerization of EpoR intracellular domain). The myristoylation signal sequence (Myr) is anchored to the amino terminal of idEpoRic for membrane association with myristate. The EpoR intracellular domain (ic, amino acids 247–406), which contains 2 tyrosine residues (Y), was fused with GFP to monitor the transgene expression. Two copies of the CID binding domain (DmrB) were inserted. The terms ec and tm indicate the extracellular domain and transmembrane domain of EpoR, respectively. (B) Sequence alignment of the mouse (amino acids 247–406) and human EpoR sequences (amino acids 248–407). Identical amino acids between the mouse (<i>M</i>) and human (<i>H</i>) EpoRs are indicated by asterisks (*). Important motifs for EpoR signaling (JM, juxtamembrane domain; Box1 and Box2, highly conserved sequence among cytokine receptors) and the phosphorylated tyrosine residues (Y343 and Y401) on mouse EpoR are illustrated. (C) Semi-quantitative RT-PCR analysis of the transgene expression in the spleens of 3 independent transgenic mouse lines. Expression of the <i>G1HRD-idEpoRic</i> transgene was detected by 27, 30, 33, 36, and 39 PCR cycles (left to right). HPRT was used as an internal control. (D) Membrane-associated localization of idEpoRic was observed in the spleen section of the line A transgenic mouse using laser confocal microscopy (left). A spleen section of a <i>G1HRD-GFP</i> transgenic mouse, which expresses GFP throughout the whole cell, is also shown as a control (right). The scale bar indicates 10 μm.