A novel distal convoluted tubule-specific Cre-recombinase driven by the NaCl cotransporter gene

Ryan J. Cornelius, Avika Sharma, Xiao Tong Su, Jin Jin Guo, Jill A. McMahon, David H. Ellison, Andrew P. McMahon, James A. McCormick

Research output: Contribution to journalArticle

Abstract

A novel distal convoluted tubule-specific Cre-recombinase driven by the NaCl cotransporter gene. Am J Physiol Renal Physiol 319: F423–F435, 2020. First published July 13, 2020; doi:10.1152/ajprenal.00101.2020.—Cre-lox technology has revolutionized research in renal physiology by allowing site-specific genetic recombination in individual nephron segments. The distal convoluted tubule (DCT), consisting of distinct early (DCT1) and late (DCT2) segments, plays a central role in Na+ and K+ homeostasis. The only established Cre line targeting the DCT is Pvalb-Cre, which is limited by noninducibility, activity along DCT1 only, and activity in neurons. Here, we report the characterization of the first Cre line specific to the entire DCT. CRISPR/Cas9 targeting was used to introduce a tamoxifen-inducible IRES-CreERT2 cassette downstream of the coding region of the Slc12a3 gene encoding the NaCl cotransporter (NCC). The resulting Slc12a3-CreERT2 mice were crossed with R26R-YFP reporter mice, which revealed minimal leakiness with 6.3% of NCC-positive cells expressing yellow fluorescent protein (YFP) in the absence of tamoxifen. After tamoxifen injection, YFP expression was observed in 91.2% of NCC-positive cells and only in NCC-positive cells, revealing high recombination efficiency and DCT specificity. Crossing to R26RTdTomato mice revealed higher leakiness (64.5%), suggesting differential sensitivity of the floxed site. Western blot analysis revealed no differences in abundances of total NCC or the active phosphorylated form of NCC in Slc12a3-Cre-ERT2 mice of either sex compared with controls. Plasma K+ and Mg2+ concentrations and thiazide-sensitive Na+ and K+ excretion did not differ in Slc12a3-Cre-ERT2 mice compared with controls when sex matched. These data suggest genetic modification had no obvious effect on NCC function. Slc12a3-CreERT2 mice are the first line generated demonstrating inducible Cre recombinase activity along the entire DCT and will be a useful tool to study DCT function.

Original languageEnglish (US)
Pages (from-to)F423-F435
JournalAmerican Journal of Physiology - Renal Physiology
Volume319
Issue number3
DOIs
StatePublished - Aug 2020

Keywords

  • Cre recombinase
  • Distal convoluted tubule
  • NaCl cotransporter
  • Potassium
  • Sodium

ASJC Scopus subject areas

  • Physiology
  • Urology

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