Targeting of PR^2VR to mCG locus. (A) A targeting vector containing the PR cDNA with the V420 and V432R mutations (PR^2VR, with position of mutations designated by asterisk) and a PGK-neo selectable marker cassette flanked by targeting arms from the mCG locus was transfected into an embryonic stem cell line (RW4; 129/SvJ). The 5 exons of the endogenous mCG gene are represented by black (coding regions) and gray (5′ and 3′ untranslated regions) rectangles. The PGK-neo^r cassette was flanked by loxP sites (gray triangles), which allowed its removal by transient transfection of ES cells with a Cre recombinase expression vector. (B) Genotyping of parental untargeted RW4 ES cells and 2 independently targeted mCG-PR^2VR clones (#1 and #7) is shown. mCG locus targeting was identified by PCR using a 3-primer multiplex cocktail (A/B/C). The untargeted mCG allele is identified by a 2.4-kb product of the forward “A” primer and a common reverse “B” primer (external to the targeting construct). The correctly targeted mCG-PR^2VR alleles are recognized as a 2.0-kb product of the forward “C” primer (within the PGK-neo cassette) and the reverse “B” primer. (C) Removal of the PGK-neo cassette was indicated by disappearance of a 1850-bp band and appearance of a 280-bp band identified by PCR (using primers “D” and “E”). 2 independent targeted mCG-PR^2VR parental clones (#1 and #7), and 4 subclones (##1-6, 1-12, 7-2, and 7-4) that have undergone successful excision of the PGK-neo^r cassette are shown. Mice generated from 2 ES clones containing independent Cre excision events (7-2 and 7-4) were used for all subsequent experiments.