Characterization of two rat models of cystic fibrosis-KO and F508del CFTR-Generated by Crispr-Cas9
Characterization of two rat models of cystic fibrosis-KO and F508del CFTR-Generated by Crispr-Cas9
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DOI：
10.1002/ame2.12091
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发表时间：
2019-12-01
影响因子：
3.7
通讯作者：
Cottart, Charles-Henry
中科院分区：
文献类型：
--
作者：
Dreano, Elise;Bacchetta, Marc;Cottart, Charles-Henry
背景基因工程动物对于正确了解囊性纤维化（CF）的疾病机制至关重要。由于其动物学能力、大小和气道特征，包括粘膜下腺体的存在，该大鼠是 CF 的相关实验室模型。方法我们描述了跨膜电导调节器 (Cftr) 基因中 p.Phe508del 突变纯合子 CF 大鼠模型 (F508del) 的生成。将该模型与新的 Cftr(-/-) 大鼠 (CFTR KO) 进行比较。通过组织切片的组织学染色检查 CF 中的靶器官，并通过微型计算机断层扫描对牙釉质进行量化。 CFTR 的活性通过鼻电位差 (NPD) 和短路电流测量来评估。分析了 VX-809 和 VX-770 对 F508del 大鼠鼻上皮原代细胞培养物的影响。结果新生 F508del 和敲除 (KO) 动物均出现肠梗阻，特殊饮食结合渗透性泻药可部分补偿肠梗阻。两种大鼠模型表现出 CF 表型异常，例如输精管发育不全和牙釉质缺陷。肠、胰腺、肝脏和肺的组织学正常。 KO大鼠中CFTR功能的缺失通过对结肠粘膜的短路电流测量进行离体证实，并通过NPD在体内进行体内证实，而在F508del大鼠中观察到残留的CFTR活性。将F508del CFTR鼻原代培养物暴露于VX-809和VX-770的组合改善了CFTR介导的Cl-转运。结论F508del大鼠再现了在CFTR KO动物中观察到的表型，并且代表了促进CF疗法开发的新资源。
BackgroundGenetically engineered animals are essential for gaining a proper understanding of the disease mechanisms of cystic fibrosis (CF). The rat is a relevant laboratory model for CF because of its zootechnical capacity, size, and airway characteristics, including the presence of submucosal glands.MethodsWe describe the generation of a CF rat model (F508del) homozygous for the p.Phe508del mutation in the transmembrane conductance regulator (Cftr) gene. This model was compared to new Cftr(-/-) rats (CFTR KO). Target organs in CF were examined by histological staining of tissue sections and tooth enamel was quantified by micro-computed tomography. The activity of CFTR was evaluated by nasal potential difference (NPD) and short-circuit current measurements. The effect of VX-809 and VX-770 was analyzed on nasal epithelial primary cell cultures from F508del rats.ResultsBoth newborn F508del and Knock out (KO) animals developed intestinal obstruction that could be partly compensated by special diet combined with an osmotic laxative. The two rat models exhibited CF phenotypic anomalies such as vas deferens agenesis and tooth enamel defects. Histology of the intestine, pancreas, liver, and lungs was normal. Absence of CFTR function in KO rats was confirmed ex vivo by short-circuit current measurements on colon mucosae and in vivo by NPD, whereas residual CFTR activity was observed in F508del rats. Exposure of F508del CFTR nasal primary cultures to a combination of VX-809 and VX-770 improved CFTR-mediated Cl- transport.ConclusionsThe F508del rats reproduce the phenotypes observed in CFTR KO animals and represent a novel resource to advance the development of CF therapeutics.