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

A HaloTag knock-in resource allows direct visualization of endogenous polycystin-2 (PC2), enabling quantitative analysis of its localization, turnover, and transport dynamics. PC2-HaloTag labeling establishes PC1-dependent and Tulp3-dependent control of PC2 ciliary targeting in vivo and in cells.

Polycystin-2, encoded by PKD2 , is a cation channel essential for kidney physiology. Dysfunction of Pkd2 causes autosomal dominant polycystic kidney disease. Currently, our understanding of cystogenesis in the kidney is limited by the difficulty of visualizing the localization and molecular functions of endogenous polycystin proteins.

Using clustered regularly interspaced short palindromic repeats/Cas9, we engineered a Pkd2 HaloTag knock-in mouse (referred to as Pkd2c-Halo ) and derived tsSV40-immortalized Pkd2c-Halo renal epithelial cell lines. We optimized HaloTag labeling for in vivo and in vitro applications, demonstrating 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 Tulp3R400W/R400W and Pkd1null mutant backgrounds.

Homozygous Pkd2c-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 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 Tulp3R400W , an allele identified in a patient with hepatorenal cystic disease, Pkd2-c-Halo failed to localize in cilia in kidney tubule epithelium.

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.