Fig. 2.

Generation and characterization of RAR-related orphan receptor C (Rorc) mutant rat. A: CRISPR/Cas9 was used to introduce a mutation in the rat Rorc gene of LEW/NCrl rat embryos by targeting the region shown in red of sequence XM_032897831.1. Gene sequencing identified an 8-bp deletion at nt 177–184. The resultant rat is referred to as LEW-Rorc^em3Mcwi or hereafter in this report as Rorc ^−/−. B: the sequence predicted a full-length protein of 508 amino acids in Rorc^+/+ rats, while the sequence in Rorc^−/− rats predicted a missense mutation after amino acid 31, leading to premature truncation. The highlighted red box illustrates the area of missense relative to wild-type protein. C: genotyping of rats following PCR amplification and BglII digestion generated fragments in Rorc^+/+ that are absent in Rorc ^−/−. D: Western blot using anti-RAR-related orphan receptor-γ (RORγT) identified a 55–60-kDa protein in the thymus from Rorc^+/+ rats, which is not present in Rorc^−/− rats. E: IL-17 mRNA expression in isolated CD4⁺ cells from the kidney following in vitro stimulation with elevated extracellular Na⁺ (170 mM) and ANG II (10⁻⁷ M). CD4⁺ cells were isolated from the kidney 7 days following ischemia-reperfusion (I/R) injury or sham from either Rorc^+/+ or Rorc^−/− rats. Data are normalized means ± SE of IL-17 mRNA, N = 3 rats per group, *P < 0.05 vs. sham, †P < 0.05 Rorc^−/− vs. Rorc^+/+ by one-way ANOVA and the Student–Newman–Keuls post hoc test.