Generation of mouse model of TGFBI-R124C corneal dystrophy using CRISPR/Cas9-mediated homology-directed repair
Generation of mouse model of TGFBI-R124C corneal dystrophy using CRISPR/Cas9-mediated homology-directed repair
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DOI：
10.1038/s41598-020-58876-w
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发表时间：
2020-02-06
影响因子：
4.6
通讯作者：
Ouchi, Yasuo
中科院分区：
文献类型：
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作者：
Kitamoto, Kohdai;Taketani, Yukako;Ouchi, Yasuo
转化生长因子-β 诱导 (TGFBI) 基因的突变会导致临床上不同类型的角膜营养不良。为了描述导致这些营养不良的机制，我们重点研究了 TGFBI 中导致格子状角膜营养不良 1 型 (LCD1) 的 R124C 突变，并使用 CRISPR/Cas9 技术通过 ssODN 介导的碱基对替换，生成了新型转基因小鼠，其中 TGFBI 中的精氨酸 124 被半胱氨酸单氨基酸替换。 80% 的纯合 TGFBI-R124C 小鼠和 9.1% 的杂合 TGFBI-R124C 小鼠在 40 周龄时出现角膜混浊。苏木精和伊红以及马森三色染色显示角膜上皮下基质中有嗜酸性沉积物，刚果红染色呈阴性。尽管在TGFBI-R124C小鼠中没有观察到淀粉样蛋白沉积，但通过透射电子显微镜在基底膜附近清楚地观察到不规则的无定形沉积物。有趣的是，我们发现纯合 TGFBI-R124C 小鼠中 TGFBI 蛋白（TGFBIp）的角膜沉积显着增加，表明突变蛋白积累具有致病作用。此外，正如在 LCD1 患者中观察到的，TGFBI-R124C 小鼠的角膜上皮伤口愈合显着延迟。总之，我们的新型 TGFBI-R124C 角膜营养不良小鼠模型再现了人类疾病的特征。该小鼠模型将有助于描述人类角膜营养不良的致病机制。
Mutations in transforming growth factor-beta-induced (TGFBI) gene cause clinically distinct types of corneal dystrophies. To delineate the mechanisms driving these dystrophies, we focused on the R124C mutation in TGFBI that causes lattice corneal dystrophy type1 (LCD1) and generated novel transgenic mice harbouring a single amino acid substitution of arginine 124 with cysteine in TGFBI via ssODN-mediated base-pair substitution using CRISPR/Cas9 technology. Eighty percent of homozygous and 9.1% of heterozygous TGFBI-R124C mice developed a corneal opacity at 40 weeks of age. Hematoxylin and eosin and Masson trichrome staining showed eosinophilic deposits in subepithelial corneal stroma that stained negative for Congo-red. Although amyloid deposition was not observed in TGFBI-R124C mice, irregular amorphous deposits were clearly observed via transmission electron microscopy near the basement membrane. Interestingly, we found that the corneal deposition of TGFBI protein (TGFBIp) was significantly increased in homozygous TGFBI-R124C mice, suggesting a pathogenic role for the mutant protein accumulation. Furthermore, as observed in the LCD1 patients, corneal epithelial wound healing was significantly delayed in TGFBI-R124C mice. In conclusion, our novel mouse model of TGFBI-R124C corneal dystrophy reproduces features of the human disease. This mouse model will help delineate the pathogenic mechanisms of human corneal dystrophy.