A HaloTag Knock-In Resource for In Vivo and In Vitro Analysis of Endogenous Polycystin-2 Localization, Turnover, and Transport

Background: Polycystin-2, encoded by PKD2 , is a cation channel essential for kidney physiology. Dysfunction of Pkd2 causes Autosomal Dominant Polycystic Kidney Disease (ADPKD). Currently our understanding of cystogenesis in the kidney is limited by the difficulty of visualizing the localization and molecular functions of endogenous polycystin proteins. Methods: Using CRISPR/Cas9, we engineered a Pkd2 HaloTag knock-in mouse (referred to as Pkd2 c-Halo ) and derived tsSV40-immortalized Pkd2 c-Halo renal epithelial cell lines. We optimized HaloTag labeling for in vivo and in vitro applications, demonstrated its use in confocal and live cell microscopy, pulse-chase assays, affinity isolation, and in vivo imaging of Pkd2 protein localization after kidney injury and in disease-relevant Tulp3 R400W/R400W and Pkd1 null mutant backgrounds. Results: Homozygous Pkd2 c-Halo mice were viable, fertile, and phenotypically normal, confirming that the C-terminal HaloTag did not disrupt Pkd2 function. Pkd2-c-Halo was detected by Western blotting and localized to endoplasmic reticulum (ER) and the primary cilium. Labeling occurred within 30 minutes and plateaued by 3 hours at doses of ≥2 nmol/mouse and ≥25 nM in vivo and in cultured cells, respectively. Pulse-chase analysis showed complete Pkd2-c-Halo turnover within 48 hours in the cilia of the choroid plexus in vivo and 24 hours in cultured renal epithelial cells. HaloTrap affinity resin purified the endogenous Pkd2-c-Halo from cells and tissues efficiently for protein complex analysis. While kidney injury is known to accelerate cyst formation, unilateral ureteral obstruction did not alter Pkd2-c-Halo expression or distribution. Finally, in a Pkd1 null background or in mice homozygous for Tulp3 R400W , an allele identified in a patient with hepatorenal cystic disease, Pkd2-c-Halo failed to localize in cilia in kidney tubule epithelium. Conclusions: The Pkd2-c-Halo mouse and derived renal epithelial cell lines enable detection of endogenous polycystin-2 in vivo and in vitro , allowing analysis of its localization, turnover under homeostatic conditions, after kidney injury, and in disease-relevant genetic backgrounds.