Structure and tissue distribution of the Epo-GFP transgene. (A) Strategy for constructing the Epo-GFP transgenes in the BAC recombination system. Epo-60K/BAC (top), including the 60-kb 5′ upstream and 120-kb 3′ downstream regions of the mouse Epo gene, was isolated. Exons I to V of the Epo gene are depicted by black boxes. The targeting vector contained GFP cDNA (white box) and a polyadenylation signal (pA; hatched box) and a Neo cassette (speckled box) between the FRT sequences (white ovals). The vector was homologously recombined with Epo-60K/BAC within the 5′ (1.1 kb) and 3′ (0.9 kb) homologous arms. The Neo cassette in the targeted BAC clone was excised using the FLP-FRT system in bacteria. To verify integration of the intact transgene into the mouse chromosome, 5′ and 3′ fragments derived from the BAC vector (a and b) and GFP cDNA (c) were amplified by PCR using primers indicated by the double arrowheads. (B) Expression of the endogenous Epo gene (bar chart) and wt-Epo-GFP transgene (panels) in adult mice. Epo mRNA levels under normal (gray bars) and anemic (black bars) conditions were measured by quantitative RT-PCR for the organs indicated and normalized to the level of GAPDH mRNA (bar chart). Data are the means (± SD) of at least 3 independent mice. Semiquantitative RT-PCR analysis of transgene expression in the wt-Epo-GFP transgenic mouse (line WA) under normal and anemic conditions was performed for the organs indicated (panels). HPRT was used as an internal control. (C) Southern blotting analysis of ApaI-digested genomic DNA revealed the copy numbers of the transgenes. Tail DNA from each transgenic line was digested with ApaI (Ap; indicated in A) and the endogenous Epo gene (5.2 kb) and transgene (4.4 kb) were hybridized with a radiolabeled probe (indicated in A).