Generation of WNK1 knockout cell lines by CRISPR/Cas-mediated genome editing.

Generation of WNK1 knockout cell lines by CRISPR/Cas-mediated genome editing. Am J Physiol Renal Physiol. 2014 Dec 4;:ajprenal.00612.2014 Authors: Roy A, Goodman JH, Begum G, Donnelly BF, Pittman G, Weinman EJ, Sun D, Subramanya AR Abstract Sodium-coupled SLC12 cation chloride cotransporters play important roles in cell volume and chloride homeostasis, epithelial fluid secretion, and renal tubular salt reabsorption. These cotransporters are phosphorylated and activated indirectly by With-No-Lysine (WNK) kinases through their downstream effector kinases, SPAK and OSR1. Multiple WNK kinases can coexist within a single cell type, although their relative contributions to SPAK/OSR1 activation and salt transport remain incompletely understood. Deletion of specific WNKs from cells that natively express a functional WNK-SPAK/OSR1 network will help resolve these knowledge gaps. Here, we outline a simple method to selectively knock out full length WNK1 expression from mammalian cells using RNA-guided CRISPR/Cas9 endonucleases. Two clonal cell lines were generated by using a single guide RNA (sgRNA) targeting exon 1 of the WNK1 gene, which produced indels that abolished WNK1 protein expression. Both cell lines exhibited reduced endogenous WNK4 protein abundance, indicating that WNK1 is required for WNK4 stability. Consistent with an on-target effect, the reduced WNK4 abundance was associated with increased expression of the KLHL3/Cullin-3 E3 ub...
Source: Am J Physiol Renal P... - Category: Urology & Nephrology Authors: Tags: Am J Physiol Renal Physiol Source Type: research