通过双 sgRNAs/Cas9 系统生成 Ext1 基因编辑小鼠模型并进行表型分析

IF 2.4 4区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY
Lan Zhou, Xin Li, Zihan Ji, Can Zhou, Lingling Yang, Yong Li, Can Fu, Lantao Gu, Shun Zhang, JinTao Gao, Pengpeng Yue, Honghao Yu
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引用次数: 0

摘要

遗传性多发性外生骨质疏松症(HME)是一种常染色体显性骨骼疾病。遗传连锁分析发现,外骨质素糖基转移酶(EXT)1和EXT2基因突变与HME发病有关,其中EXT1基因突变最为常见。本研究的目的是利用Ext1基因编辑技术模拟人类Ext1基因突变,建立小鼠模型,并通过表型分析研究Ext1基因的致病性。我们设计了一对针对小鼠Ext1基因第1外显子的双sgRNA,以精确删除46 bp的DNA片段,从而导致Ext1基因的框移突变。将设计好的双 sgRNAs 和 Cas9 蛋白注入小鼠子代细胞质中。通过胚胎移植共获得14只小鼠,其中两只基因型嵌合体小鼠的Ext1基因外显子1的46 bp DNA片段缺失。通过杂交和育种,我们成功培育出了Ext1基因被编辑的杂合子小鼠(Ext+/-)。脱靶效应分析没有发现所使用的两种 sgRNA 在 Ext+/- 小鼠中引起脱靶突变。与野生型小鼠相比,Ext+/-小鼠的体重较轻。X射线成像显示,只有雄性Ext+/-小鼠尾椎附近的骨质增生,肋骨和脊柱区域之间骨质增生的计算机断层扫描值约为200 HU。此外,免疫组化分析显示,与野生型小鼠相比,Ext+/-小鼠中表达EXT1的关节软骨细胞较少。病理切片分析表明,Ext+/-小鼠的心脏、肝脏、肺或肾脏组织没有结构或形态异常。总之,我们利用sgRNAs/Cas9双系统成功地生成了一个精确的DNA缺失模型,用于研究Ext1。总之,我们利用双 sgRNAs/Cas9 系统成功地在 Ext1 小鼠模型中产生了精确的 DNA 缺失。总之,我们在 Ext+/- 小鼠中观察到了明显的表型变化,尤其是雄性个体的骨质增生;但是,在基因编辑的小鼠中没有检测到骨质疏松症。通过 CRISPR/Cas9 技术在 Ext1 基因中引入框移突变导致了新的表型改变,突出了 Ext1 的遗传致病性。因此,我们的 Ext+/- 小鼠是进一步开展 Ext1 基因相关生物医学研究的宝贵模型。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Generation of Ext1 Gene-Edited Mice Model Via Dual sgRNAs/Cas9 System and Phenotypic Analyses.

Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disease. Genetic linkage analyses have identified that mutations in the exostosin glycosyltransferase (EXT)1 and EXT2 genes are linked to HME pathogenesis, with EXT1 mutation being the most frequent. The aim of this study was to generate a mice model with Ext1 gene editing to simulate human EXT1 mutation and investigate the genetic pathogenicity of Ext1 through phenotypic analyses. We designed a pair of dual sgRNAs targeting exon 1 of the mice Ext1 gene for precise deletion of a 46 bp DNA fragment, resulting in frameshift mutation of the Ext1 gene. The designed dual sgRNAs and Cas9 proteins were injected into mice zygotes cytoplasm. A total of 14 mice were obtained via embryo transfer, among which two genotypic chimera mice had a deletion of the 46 bp DNA fragment in exon 1 of the Ext1 gene. By hybridization and breeding, we successfully generated heterozygous mice with edited Ext1 gene (Ext+/-). Off-target effect analysis did not reveal off-target mutations in Ext+/- mice caused by the two sgRNAs used. Compared to wild-type mice, Ext+/- mice exhibited lower body weights. X-ray imaging showed hyperplastic bone near caudal vertebrae only in male Ext+/- mice, with computed tomography values approximately at 200 HU for hyperplastic bone between ribs and spine regions. Furthermore, immunohistochemical analysis revealed fewer articular chondrocytes expressing EXT1 in edited mice compared to wild-type ones. Pathological section analysis demonstrated no structural or morphological abnormalities in heart, liver, lung, or kidney tissues from Ext+/- mice. In conclusion, we successfully generated an accurate DNA deletion model for studying Ext1 using dual sgRNAs/Cas9 systems. In conclusion, we successfully generated precise DNA deletions in the Ext1 mice model using the dual sgRNAs/Cas9 system. In conclusion, we observed significant phenotypic changes in Ext+/- mice, particularly bone hyperplasia in male individuals; however, no exostosis was detected in the gene-edited mice. The introduction of a frameshift mutation into the Ext1 gene through CRISPR/Cas9 technology resulted in novel phenotypic alterations, highlighting the genetic pathogenicity of Ext1. Therefore, our Ext+/- mice serve as a valuable model for further biomedical investigations related to the Ext1 gene.

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来源期刊
Molecular Biotechnology
Molecular Biotechnology 医学-生化与分子生物学
CiteScore
4.10
自引率
3.80%
发文量
165
审稿时长
6 months
期刊介绍: Molecular Biotechnology publishes original research papers on the application of molecular biology to both basic and applied research in the field of biotechnology. Particular areas of interest include the following: stability and expression of cloned gene products, cell transformation, gene cloning systems and the production of recombinant proteins, protein purification and analysis, transgenic species, developmental biology, mutation analysis, the applications of DNA fingerprinting, RNA interference, and PCR technology, microarray technology, proteomics, mass spectrometry, bioinformatics, plant molecular biology, microbial genetics, gene probes and the diagnosis of disease, pharmaceutical and health care products, therapeutic agents, vaccines, gene targeting, gene therapy, stem cell technology and tissue engineering, antisense technology, protein engineering and enzyme technology, monoclonal antibodies, glycobiology and glycomics, and agricultural biotechnology.
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