Targeting PML-RARα to the murine cathepsin G locus by homologous recombination and generation of transgenic mice. (A) A targeting vector consisting of a PML-RARα cDNA and PGK-neo selectable marker cassette, flanked by targeting arms from the murine cathepsin G (mCG) locus were transfected into embryonic stem cells. Coding exons of the mCG gene are represented as black boxes (not to scale). The PML-RARα cDNA and PGK-neo selectable marker cassette are represented as open boxes. Transcriptional start sites for the mCG gene and PGK-neo selectable marker cassette are indicated by arrows. The mCG 5′ untranslated region is represented by a gray box. LoxP sites used for CRE recombinase-mediated cassette excision are represented as shaded ovals. The position of an external 3′-mCG DNA probe is indicated as a hatched box under mCG exons 3 and 4. The PGK-neo cassette was subsequently removed from a homologously recombinant ES cell clone by transient transfection of targeted ES cells with a Cre recombinase expression vector. (B) The targeted mCG locus was identified in ES cells by a size shift from 3.2 kb (wild type [WT]) to 7.9 kb (targeted, +PGK-neo) following HindIII digestion of genomic DNA. Removal of the PGK-neo cassette by Cre recombinase resulted in a shift in the size of the recombinant band to 6.4 kb (a loading artifact caused the slight apparent differences in the sizes of the WT mCG bands at 3.2 kb). (C) Tail DNA from the offspring a heterozygous CG^PR/+ intercross was screened by Southern blotting to identify wild-type (CG^+/+), heterozygous (CG^+/PR), and homozygous (CG^PR/PR) animals. (D) Tail DNA from the offspring of CG^PR/PR × CG^+/– animals was screened by Southern blotting to identify animals that carried one PML-RARα allele together with either an intact CG gene (CG^+/PR) or a CG null mutation (CG^–/PR) at the other allele.