| Kcnh6点突变单基因糖尿病家系的小鼠模型构建及表型分析 |
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| 引用本文： | Kcnh点突变单基因糖尿病家系的小鼠模型构建及表型分析.Kcnh6点突变单基因糖尿病家系的小鼠模型构建及表型分析[J].首都医学院学报,2021,42(4):575-581. |
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| 作者姓名： | Kcnh点突变单基因糖尿病家系的小鼠模型构建及表型分析 |
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| 作者单位： | 首都医科大学附属北京同仁医院内分泌科 糖尿病防治研究北京市重点实验室 北京市糖尿病研究所,北京 100730 |
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| 基金项目： | 国家自然科学基金(81800688, 82070890, 81930019),国家重点研发计划(2017YFC0909600),北京市属医院科研培育计划(PX2019006)。 |
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| 摘 要： | 目的 通过CRISPR/Cas9技术构建Kcnh6基因P235L位点基因敲入定点突变小鼠模型,并验证该突变能否成功复制人类糖尿病家系表型,为接下来研究Kcnh6基因在糖尿病发病中的作用提供更精确的实验工具。方法 设计针对已知糖尿病家系点突变位点的sgRNA(small guide RNA,sgRNA),同时制作一个携带靶位点同源臂、突变位点序列的供体DNA敲入载体。将sgRNA、cas9 mRNA和敲入载体通过显微注射移入小鼠的受精卵,选取其中存活的受精卵将其移植入假孕小鼠子宫,繁育得到子代小鼠。采用聚合酶链反应和基因测序等方法检测子代小鼠Kcnh6基因碱基突变情况。监测基因敲入小鼠的体质量和并对小鼠做糖耐量和胰岛素分泌实验。结果 成功构建Kcnh6-P235L位点基因敲入纯合子小鼠。在高脂饲料喂养条件下,与同窝野生型(wild type, WT)小鼠相比,Kcnh6 基因插入(knock in, KI)点突变小鼠的体质量增加,糖耐量及胰岛素分泌功能受损。结论 成功构建了Kcnh6基因点突变敲入小鼠模型,且此模型具有糖尿病相关表型。
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| 关 键 词： | CRISPR/Cas9 定点突变 Kcnh6基因 糖耐量异常 |
| 收稿时间： | 2021-01-21 |
Establishment of Kcnh6 gene site-specific mutant mice modeled on a pedigree with monogenic diabetes and the phenotype analysis |
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| Xiong Fengran,Lu Jing,Zhang Yingchao,Xie Rongrong,Zhao Ruxuan,Li Qi,Yang Jinkui.Establishment of Kcnh6 gene site-specific mutant mice modeled on a pedigree with monogenic diabetes and the phenotype analysis[J].Journal of Capital University of Medical Sciences,2021,42(4):575-581. |
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| Authors: | Xiong Fengran Lu Jing Zhang Yingchao Xie Rongrong Zhao Ruxuan Li Qi Yang Jinkui |
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| Affiliation: | Endocrinology Department of Beijing Tongren Hospital, Capital Medical University, Beijing Key Laboratory of Diabetes Research and Care, Bejing Diabetes Institute, Beijing 100730, China |
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| Abstract: | Objective To establish the Kcnh6 gene P235L site-specific mutant mice model by using CRISPR/Cas9 technique and to verify whether the phenotype is consistent with the diabetes pedigree that we found previously,in order to provide a more precise experimental tool for studying the crucial role of Kcnh6 gene in the pathogenesis of diabetes. Methods sgRNA specific for the point-mutant site was designed according to the exon 5 of Kcnh6 gene. Then,we constructed the donor vector containing the homologous arm and mutant site sequence based on the sequence of the sgRNA. Finally,sgRNA,donor vector and Cas9 were mixed together and microinjected into fertilized eggs of mice.The survived oosperms were transplanted into the uteruses of pseudopregnant mice.We detected the Kcnh6 gene mutation of the progeny by PCR and gene sequencing technology. Results We established the homozygous Kcnh6 gene P235L site-specific mutant mice. Preliminary studies showed that Kcnh6 gene knock-in site-specific mutant mice have less body weight,impaired glucose tolerance and decreased insulin secretion by feeding high fat diet,compared to wild-type mice. Conclusion We successfully constructed the homozygous Kcnh6 gene P235L site-specific mutant mice model which will provide a more precise research tool to exploit the crucial role of Kcnh6 gene in the pathogenesis of diabetes. |
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| Keywords: | CRISPR/Cas9 site-specific mutation Kcnh6 gene impaired glucose tolerance |
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