1. Generation of Alpha-1 Antitrypsin Knockout and PI*ZZ Ferrets Using Crispr/Cas9. A Genetic Model of Emphysema
- Author
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Marina Zieger, Jaimie S. Gray, Bradley H. Rosen, Nan He, Idil A. Evans, John F. Engelhardt, Shashanna R. Moll, Michael H. Brodsky, Xingshen Sun, Christian Mueller, Florie Borel, Bo Liang, and Ziying Yan
- Subjects
0301 basic medicine ,Pulmonary and Respiratory Medicine ,business.industry ,Alpha (ethology) ,Pulmonary disease ,respiratory system ,Abstracts ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,030228 respiratory system ,Immunology ,Genetic model ,Pi ,Medicine ,CRISPR ,business - Abstract
Rationale: The most prevalent genetic cause of chronic obstructive pulmonary disease is alpha-1 antitrypsin (A1AT) deficiency, a disorder that has yet to be widely modeled in animals because of species-specific differences between rodents and humans. Objectives: To address these challenges, we engineered two A1AT ferret models using zygote gene editing to test the hypothesis that unopposed protease activity within the lung leads to emphysema and bronchitis. Methods: Guide RNAs targeting exon 2 (for knockout) and exon 5 (for Z-allele mutation, Pi*Z) of the ferret A1AT gene were injected into ferret zygotes with Cas9 mRNA. For PI*Z targeting, a short oligonucleotide carrying the mutation was included. Offspring were genotyped and plasma levels of A1AT determined by Western blot before entry into a longitudinal study. Adult A1AT ferrets underwent bronchoscopy and FlexiVent over time to characterize lung disease. Control animals were wild-type (WT) and age, sex, and size matched. Results: A1AT-deficient ferrets spontaneously develop hallmarks of emphysema as early as 3 months when compared with matched WT control ferrets. Over time, A1AT-KO ferrets reached an inspiratory capacity of 117% ± 5% (WT, 57.4 ml; A1AT, 67.5 ml) and compliance of 111% ± 4% (WT, 5.5 ml/cm H(2)O; A1AT, 6.1 ml/cm H(2)O; N = 8 pairs A1AT/WT, P
- Published
- 2018