| Title: |
A HaloTag Knock-In Resource for In Vivo and In Vitro Analysis of Endogenous Polycystin-2 Localization, Turnover, and Transport |
| Authors: |
Li, Zhang; Haycraft, Courtney J.; Croyle, Mandy J.; Hudson, Daniel; Yuan, Yuan; Simanyi, Kristin; Vendrame, Hanan Chweih; Wang, Jun; Kachwala, Alfiya Ibrahimbhai; Ma, Yongjie; Parant, John M.; Chumley, Phillip; Zhou, Juling; Mrug, Michal; Parnell, Stephen C.; Tran, Pamela V.; Gao, Hongjuan; Qian, Feng; Outeda, Patricia; Wallace, Darren P.; Watnick, Terry J.; Yoder, Bradley K. |
| Contributors: |
National Institute of Diabetes and Digestive and Kidney Diseases; National Institute of Child Health and Human Development; US Department of Veterans Affairs |
| Source: |
Journal of the American Society of Nephrology ; ISSN 1046-6673 1533-3450 |
| Publisher Information: |
Ovid Technologies (Wolters Kluwer Health) |
| Publication Year: |
2026 |
| Description: |
Key Points 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. Background 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. Methods Using clustered regularly interspaced short palindromic repeats/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, 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 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 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, ... |
| Document Type: |
article in journal/newspaper |
| Language: |
English |
| DOI: |
10.1681/asn.0000001036 |
| DOI: |
10.1681/ASN.0000001036 |
| Availability: |
https://doi.org/10.1681/asn.0000001036; https://journals.lww.com/10.1681/ASN.0000001036 |
| Rights: |
http://creativecommons.org/licenses/by/4.0/ |
| Accession Number: |
edsbas.F7EF8332 |
| Database: |
BASE |