(A) Structure of the Ptpn11 targeting locus and targeting construct region. Protein coding regions, LoxP sites, and Frt sites are shown. The arrows highlight the target positions of PCR primers used to identify homologous recombinants. Note that primers PLF and PRR are outside the region of homology. The expected amplicons are indicated with brackets and are specific to homologously recombined templates. LoxP sites are shown as black triangles; mutant LoxP511 sites are shown as white triangles. Their relative orientation is shown by the orientation of the triangles. FRT sites are shown as white ovals. Exons are indicated by rectangles. (B) Structure of locus after Flp-mediated recombination. (C) Structure of locus after Cre-mediated recombination. (D) Confirmation of correct targeting of ES cell clone 1F10 by PCR, revealing a 4.6-kb fragment on each correctly targeted side of the allele. M indicates molecular weight markers. (E) Germline transmission in F1 mice, as depicted by PCR, for the presence of the neo gene. (F) PCR detection strategy using LSGenofwd and LSR primers for detection of WT and LS/+ mice. (G) Restriction digest (HpyCH4V) of Ptpn11 exon 7 RT-PCR–derived fragment confirming expression of the mutant allele (full-length = 185 bp; fragment sizes = 100, 75 bp, 58 bp, 42 bp, and 10 bp [LS]; 100 bp, 75 bp, and 10 bp [WT]). Note that the 10-bp fragment probably comigrates with primer alone and/or contaminating RNA. PCR primers used are 721 forward/900 reverse. See Supplemental Table 1 for details on primers used throughout this figure. Fwd, forward; Rev, reverse.