Extended Data Fig. 1. FOXI1 deletion impairs kidney intercalated cell formation and mucociliary clearance in ferrets.
a, Schematic of the approach to generate FOXI1-KO ferrets by Cas9/sgRNA RNP injection of zygotes. Bottom, sequence of the founder indel insertions (+) and deletions (∆) with gRNA sequence (green) and PAM sequence (red) shown. b, Representative sequence chromatograms of the DNA target site from non-injected (control) and FOXI1 RNP-injected zygotes (black arrow indicates cleavage site). c, Representative TIDE analysis from a compound heterozygous FOXI1-KO (−14/−4) founder ferret showing percentage of DNA editing for each indel size. d, Predicted amino acid sequence of ferret FOXI1 in wild-type (top) and FOXI1-KO founders (bottom). Asterisks indicates a premature stop codon found in the coding sequence of FOXI1 mutants. Red letters indicate frameshift mutation caused by indels. e, Kidney tissue lysates from FOXI1-KO and WT animals were collected and subjected to Western blotting with FOXI1 antibody. Representative samples show the absence of FOXI1 protein in KO animal kidney tissues. f, RT-qPCR of kidney mRNA showing absent or reduced intercalated cell (IC) marker (SLC26A4, AE1, ATP6V0D2) expression in FOXI1-KO ferrets. Mean ± s.e.m.; n = 8 animals in each group. g, Representative 68Ga-MAA PET/CT images of FOXI1-KO and WT ferret trachea at the indicated time points showing reduced clearance in FOXI1-KO. h, Micro-optical coherence tomography (μOCT) imaging of FOXI1-KO and WT ferret tracheal explants. Images show airway surface liquid (ASL; yellow bar), mucus layer (mu), and periciliary liquid (PCL; green bar) on the luminal surface. Analysis of μOCT images from explanted ferret trachea yields numerical values for functional and anatomic parameters. i,j, PCL depths (i) and ciliary beat frequency (CBF) (j) were analyzed geometrically as shown in bar graph. Mean ± s.e.m.; n = 8 animals in each group. Statistical significance was determined by: (f) two-tailed Student’s t-test and (i,j) ROI using t-tests with pooled SD by R, statistical test was two-sided.