A novel distal convoluted tubule-specific Cre-recombinase driven by the NaCl cotransporter gene
Abstract
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-Cre-ERT2 cassette downstream of the coding region of the Slc12a3 gene encoding the NaCl cotransporter (NCC). The resulting Slc12a3-Cre-ERT2 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 R26R-TdTomato 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-Cre-ERT2 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.
Funding
This work was supported by by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK120790 (to R. J. Cornelius), DK098141 and DK117903 (to J. A. McCormick), DK110792 (to A. P. McMahon), and DK054196 and DK054983 (to D. H. Ellison) as well as Veterans Affairs Grant 1I01BX002228 and a Fondation LeDucq Transatlantic Network of Excellence grant (to D. H. Ellison).
Contributions
R.J.C., J.-J.G., J.A. McMahon, A.P.M., and J.A. McCormick conceived and designed research; R.J.C., A.S., X.-T.S., J.-J.G., J.A. McMahon, and J.A. McCormick performed experiments; R.J.C., J.-J.G., J.A. McMahon, D.H.E., A.P.M., and J.A. McCormick analyzed data; R.J.C., D.H.E., A.P.M., and J.A. McCormick interpreted results of experiments; R.J.C. prepared figures; R.J.C. and J.A. McCormick drafted manuscript; R.J.C., D.H.E., A.P.M., and J.A. McCormick edited and revised manuscript; R.J.C., A.S., X.-T.S., J.-J.G., J.A. McMahon, D.H.E., A.P.M., and J.A. McCormick approved final version of manuscript.
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Additional details
- PMCID
- PMC7509282
- National Institute of Diabetes and Digestive and Kidney Diseases
- DK120790
- National Institute of Diabetes and Digestive and Kidney Diseases
- DK098141
- National Institute of Diabetes and Digestive and Kidney Diseases
- DK117903
- National Institute of Diabetes and Digestive and Kidney Diseases
- DK110792
- National Institute of Diabetes and Digestive and Kidney Diseases
- DK054196
- National Institute of Diabetes and Digestive and Kidney Diseases
- DK054983
- United States Department of Veterans Affairs
- 1I01BX002228
- Fondation Leducq
- None
- Accepted
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2020-07-02Accepted
- Available
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2020-08-28Published online
- Caltech groups
- Division of Biology and Biological Engineering
- Publication Status
- Published