Targeted insertion of the GFPcre reading frame into the EpoR locus and screening strategy. (A) Structure of the genomic and the targeted locus of the EpoR. The targeting vector is shown in the middle of panel A. The exons of the EpoR gene are numbered and shown as black boxes. The oval indicates the polyadenylation signal (pA); triangles, FRT sites; a black/dark gray box, the GFPcre sequence inserted in exon 1 forming the new exon 1*; an open box, the neo^r selection marker; and a dashed line, the vector backbone of the targeting construct. Annealing sites of external probe A (EpoR) as well as internal probe B (GFP), C (cre), and D (neo) are indicated by black lines. (B) Southern blot analysis of EcoRV-digested genomic DNA derived from Balb/c and homologous recombinant ES cell clones. A blot probed by external probe A as well as internal probes B, C, and D is shown. The 5′-EcoRV fragment of the targeted knock-in allele (ki) is represented by a 7.4-kb band, and the wild-type EpoR 5′-EcoRV fragment (WT) is represented by a 9.0-kb band (probe A, B). The 3′-EcoRV fragment of the knock-in allele carrying the FRT-flanked neo-cassette (FnFpA; clone no. 201) is displayed by a 6.9-kb fragment (probe C and D) and the same allele having eliminated the selection marker (FpA; clone no. 201-47, no. 201-91) by a 5.6-kb band (probe C). (C) Multiplex PCR analysis of ErGFPcre mice. The wild-type allele (WT) displays a 431-bp PCR product created by the primers b and c, whereas a 679-bp band represents the GFPcre knock-in (ki) allele generated with the primers a and c. The PCR method was tested on Southern blot–confirmed (bottom left) genomic tail DNA derived from male and female wild-type Balb/c or hemizygous ErGFPcre mice.