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 non-inducibility, 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 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 blotting revealed no differences in abundances of total or the active-phosphorylated form of NCC in Slc12a3-Cre-ERT2 mice of either sex compared to controls. Plasma [K⁺] and [Mg²⁺], and thiazide-sensitive Na⁺ and K⁺ excretion did not differ in Slc12a3-Cre-ERT2 mice compared to 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.

中文翻译：

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一种由 NaCl 协同转运蛋白基因驱动的新型远曲小管特异性 Cre 重组酶。

Cre-lox 技术允许在单个肾单位节段中进行位点特异性基因重组，从而彻底改变了肾脏生理学的研究。远曲小管 (DCT) 由不同的早期 (DCT1) 和晚期 (DCT2) 节段组成，在 Na ⁺和 K ^{+ 中}起核心作用体内平衡。唯一已建立的针对 DCT 的 Cre 系是 Pvalb-Cre，它受到不可诱导性、仅沿 DCT1 的活动和神经元活动的限制。在这里，我们报告了特定于整个 DCT 的第一条 Cre 线的特征。CRISPR/Cas9 靶向用于在编码 NaCl 协同转运蛋白 (NCC) 的 Slc12a3 基因编码区下游引入他莫昔芬诱导型 IRES-Cre-ERT2 盒。将得到的 Slc12a3-Cre-ERT2 小鼠与 R26R-YFP 报告小鼠杂交，在没有他莫昔芬的情况下，6.3% 的表达 YFP 的 NCC 阳性细胞泄漏最小。他莫昔芬注射后，在 91.2% 的 NCC 阳性细胞中观察到 YFP 表达，仅在 NCC 阳性细胞中观察到 YFP 表达，显示出高重组效率和 DCT 特异性。与 R26R-TdTomato 小鼠杂交显示更高的渗漏（64. 5%)，表明 floxed 位点的不同敏感性。蛋白质印迹显示，与对照相比，任一性别的 Slc12a3-Cre-ERT2 小鼠中 NCC 的总或活性磷酸化形式的丰度没有差异。等离子 [K⁺ ] 和 [Mg ²⁺ ]，以及当性别匹配时，Slc12a3-Cre-ERT2 小鼠与对照组相比，噻嗪类药物敏感的 Na ⁺和 K ⁺排泄没有差异。这些数据表明基因改造对 NCC 功能没有明显影响。Slc12a3-Cre-ERT2 小鼠是在整个 DCT 中展示可诱导 Cre 重组酶活性的第一条线，并将成为研究 DCT 功能的有用工具。