Establishment of rescued EpoR mutant mouse lines.

(A) Structure of the wild-type and mutant EpoRlocus4 and design of the HG1-EpoR andHG1-GFP transgenes. The GATA-1–HRD minigene containing exons IE and II of the mouse GATA-1 gene was ligated to either EpoR or GFP cDNA to give HG1-EpoR andHG1-GFP, respectively. The translated and untranslated exons of the EpoR gene are shown as solid and hatched boxes, respectively. Neo, pA, and Av represent the neomycin resistance gene cassette, polyadenylation signal, and AvrII sites, respectively. (B) Genotyping of transgenic and rescued mice by Southern blot analysis. Tail DNA samples digested with AvrII were hybridized to the EpoR probe shown in panel A. Tg-A (lane 1) and Tg-B (lane 2) mice contain 40 and 4 copies of theEpoR transgene, respectively. Note that the wild-typeEpoR band (4.2 kb) is absent in lanes 7 and 8 (Tg-B rescued mice), and a knockout allele band is present in lanes 4 to 8. (C) Expected mRNA structures of endogenous and transgenic EpoR. Transgene-derived EpoR mRNA includes exons IE and II of theGATA-1–HRD, so that its amplicon can be distinguished from endogenous EpoR-derived mRNA using the primer sets shown. (D) Endogenous and transgenic EpoR mRNA expression determined by RT-PCR analysis. Samples of total RNA from various tissues of wild-type (lanes 1-8), Tg-A (lanes 9-16), Tg-B (lanes 17-24), and Rescued-B (lanes 25-32) mice were analyzed. PCR was performed using primer sets Primer 1 with 2 and Primer 3 with 2 to detect endogenous (600 bp) and transgenic (701 bp) EpoR transcripts, respectively. HPRT was used as an internal control. Br indicates brain; H, heart; K, kidney; S, spleen; U, uterus; M, muscle; B, bone marrow; and T, testis.