Targeting strategy of CT-GFP inducible R26CT mouse. (A). Top: The structure of targeting vector containing a loxP-flanked (bgeo/stop) cassette with CT-GFP pA target gene. A pROSA26-1 plasmid contains ROSA26 genomic sequences and a diphtheria toxin gene (PGK-DTA) to increase rate of homologous recombination and negative selection in ES cells, respectively. Middle: the genomic organization of endogenous Rosa26 locus. The structure of the targeted Rosa26 allele (R26CT) which expresses β-galactosidase and a neomycin resistance cassette fusion gene mRNA. Bottom: Cre-mediated excision of targeted R26CT allele to transcribe CT-GFP mRNA. loxP, solid arrow head; βgeo, a fusiongene of β-galactosidase and neomycin (Neo) resistant gene;. pA, polyA signal; STOP, a transcription stop sequence. (B) Southern Blot analysis of genomic DNA prepared from Neo resistant ES cell lines. It was digested with EcoRV and hybridized with a32P-labeled 5' probe indicated in A. The 3.8 kbp EcoRV fragment indicates the targeted R26CT allele and the 11 kbp EcoRV fragment represents wild type Rosa26 allele. (C) PCR screening for the targeting event. A set of primers shown in A were used to amplify 1.2 kbp (Pa and Pb) and 0.85 kbp (Pc and Pd) size fragments from the R26CT allele, but not wild type Rosa26 allele. (D) Southern Blot analysis of genomic DNA prepared from heterozygous R26CT and wild type mice. After EcoRV digestions, the 5' probe was used the same as mentioned in A. (E) PCR screening for the targeting event to amplify 1.2 kbp band from the R26CT allele.