Phenotypes of CF rabbits generated by CRISPR/Cas9-mediated disruption of the CFTR gene
Jie Xu, … , Richard C. Boucher, Fei Sun
Jie Xu, … , Richard C. Boucher, Fei Sun
Published November 24, 2020
Citation Information: JCI Insight. 2021;6(1):e139813. https://doi.org/10.1172/jci.insight.139813.
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Research Article Cell biology Pulmonology

Phenotypes of CF rabbits generated by CRISPR/Cas9-mediated disruption of the CFTR gene

Abstract

Existing animal models of cystic fibrosis (CF) have provided key insights into CF pathogenesis but have been limited by short lifespans, absence of key phenotypes, and/or high maintenance costs. Here, we report the CRISPR/Cas9-mediated generation of CF rabbits, a model with a relatively long lifespan and affordable maintenance and care costs. CF rabbits supplemented solely with oral osmotic laxative had a median survival of approximately 40 days and died of gastrointestinal disease, but therapeutic regimens directed toward restoring gastrointestinal transit extended median survival to approximately 80 days. Surrogate markers of exocrine pancreas disorders were found in CF rabbits with declining health. CFTR expression patterns in WT rabbit airways mimicked humans, with widespread distribution in nasal respiratory and olfactory epithelia, as well as proximal and distal lower airways. CF rabbits exhibited human CF–like abnormalities in the bioelectric properties of the nasal and tracheal epithelia. No spontaneous respiratory disease was detected in young CF rabbits. However, abnormal phenotypes were observed in surviving 1-year-old CF rabbits as compared with WT littermates, and these were especially evident in the nasal respiratory and olfactory epithelium. The CF rabbit model may serve as a useful tool for understanding gut and lung CF pathogenesis and for the practical development of CF therapeutics.

Authors

Jie Xu, Alessandra Livraghi-Butrico, Xia Hou, Carthic Rajagopalan, Jifeng Zhang, Jun Song, Hong Jiang, Hong-Guang Wei, Hui Wang, Mohamad Bouhamdan, Jinxue Ruan, Dongshan Yang, Yining Qiu, Youming Xie, Ronald Barrett, Sharon McClellan, Hongmei Mou, Qingtian Wu, Xuequn Chen, Troy D. Rogers, Kristen J. Wilkinson, Rodney C. Gilmore, Charles R. Esther Jr., Khalequz Zaman, Xiubin Liang, Michael Sobolic, Linda Hazlett, Kezhong Zhang, Raymond A. Frizzell, Martina Gentzsch, Wanda K. O’Neal, Barbara R. Grubb, Y. Eugene Chen, Richard C. Boucher, Fei Sun

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Figure 6

Characterization of rabbit CFTR expression and function in the lower airways.

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Characterization of rabbit CFTR expression and function in the lower air...
(A) Representative micrographs of trachea, large bronchi, and small airways (bronchiole) from WT rabbits (approximately 3 months old), stained with H&E or probed for CFTR and negative control mRNA by RNAscope (red chromogen). Scale bar: 20 μm. n = 2 rabbits. (B) CFTR Western blots for normal human bronchial epithelial (HBE) cell cultures alongside tracheal tissue lysates from WT and CF rabbits (Δ1 line). Position of band B and C are indicated, and Western blot for β actin is shown as loading control. (C) IHC for CFTR in nasal olfactory epithelia, nasal submucosal glands, and pancreas, where CFTR (black chromogen, arrowheads) was detected in cells consistent with the distribution of ionocytes, ductal cells, and collecting ducts, respectively. Scale bar: 20 μm. (D) Gross images of tracheas isolated from WT and CF rabbits. CF tracheas lack concentric and equally spaced cartilaginous rings compared with WT rabbits. (E) Ussing chamber measurements of short-circuit currents (Isc) in freshly excised WT and CF rabbit tracheas. Isc changes in response to amiloride (amil), forskolin/IBMX (Fsk/IBMX), GlyH101 (Gly101), and bumetanide (Bumet) are shown. Basal Isc and transepithelial resistance were 49.82 ± 14.23 versus 57.74 ± 2.52 μA/cm2 and 60.07 ± 13.7 versus 60.66 ± 11.4 Ωcm2 for WT versus CF rabbits, respectively. n = 7 WT and 7 CF rabbits. *P < 0.05 versus WT rabbits. Unpaired, 2-tailed t test with Welch’s correction for unequal variance. (F and G) Ussing chamber measurements of Isc for WT and CF rabbit tracheal epithelial cell cultures (F) (WT n = 3 and CF n = 2, 3–4 inserts/code), and normal and CF human bronchial epithelial cell cultures (G) (NL [n = 5] and CF [n = 1], 1717 1-G>A/N1303K compound mutation, no CFTR protein detectable, 3–4 inserts/code), illustrating responses to forskolin (Fsk), Vertex 770 (VX770), CFTR inhibitor 172 (I-172), and uridine triphosphate (UTP). *P < 0.05 versus WT cultures. Each dot represents the average of 3–4 inserts measurements. Unpaired, 2-tailed t test with Welch’s correction for unequal variance.