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<p>A novel reporter model for <i>Vhl</i> inactivation in the mouse kidney. <b>A,</b> Design and recombination of the cell marking conditional <i>Vhl<sup>pjr</sup></i> allele. Double and single arrows indicate reversible and irreversible processes, respectively. <i>Vhl<sup>pjr.fl</sup>, Vhl<sup>pjr.inrec</sup></i>, and <i>Vhl<sup>pjr.KO</sup></i> refer to “floxed,” “incompletely recombined,” and “knockout” forms of the <i>Vhl<sup>pjr</sup></i> allele. P, <i>Vhl</i> promoter; U, untranslated region; E, <i>Vhl</i> exon; I, <i>Vhl</i> intron; pA, polyadenylation site; P2A, porcine teschovirus 2A peptide; SA, splice acceptor. Dashed lines, spliced and translated regions; lightning symbols, excitation and emission wavelengths for tdTomato fluorescence. Red stop sign indicates no interaction between VHL exon 1 fragment and HIFA-1/2 or Elongin B/C. <b>B,</b> Representative tdTomato IHC counterstained with hematoxylin in kidney sections and tdTomato fluorescence-based flow cytometry on renal cells from <i>Vhl<sup>wt/pjr.fl</sup>; Pax8-CreERT2</i> mice untreated (top) or given 5 × 2 mg tamoxifen (TMX; bottom) and harvested at the early time point. Scale bar, 250 μm. Magnification, ×20. FACS gates are shown. <b>C,</b> Gel electrophoresis of genomic PCR for <i>Vhl<sup>wt</sup>, Vhl<sup>pjr.fl</sup></i>, and <i>Vhl<sup>pjr.KO</sup></i> alleles performed on FAC-sorted tdTomato-positive (left) or tdTomato-negative (right) cells from kidneys of <i>Vhl<sup>wt/pjr.fl</sup>; Pax8-CreERT2</i> mice given tamoxifen and harvested at the early time point. <b>D,</b> Representative immunoblots (IB) for HIF1A, HIF2A, or tdTomato protein in tdTomato-negative (−) or tdTomato-positive (+) cells sorted by flow cytometry from dissociated kidneys of <i>Vhl<sup>jae.KO/pjr.fl</sup></i> or <i>Vhl<sup>wt/pjr.fl</sup> Pax8-CreERT2</i> mice given 5 × 2 mg tamoxifen and harvested at the early time point (<i>n</i> = 3 per genotype).</p>

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