基于CRISPR/Cas9联合Cre-LoxP方法的Ddx3x肝脏条件性敲除小鼠模型的构建与鉴定

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中文摘要：目的采用 CRISPR/Cas9 联合 Cre-LoxP 方法构建 DEAD-box RNA 解旋酶 3X 连锁(DEAD-box RNA helicase 3X-linked,Ddx3x)肝脏条件性敲除小鼠.方法设计特异的 sgRNA序列,在 Ddx3x基因外显子 4～10 两端插入 LoxP 序列,构建 Ddx3x-flox小鼠.采用 PCR方法对 F0 代和 F1 代小鼠进行基因鉴定.将 Ddx3x-flox F1 代小鼠与特异性表达 Alb-Cre的工具鼠交配,获得肝脏条件性敲除 Ddx3x基因小鼠(Ddx3xΔhep).采用 PCR方法和 Western blotting 方法分别检测 Ddx3xΔhep 基因小鼠目的基因与蛋白的表达.选取 8 周龄雄性野生型 C57/6J小鼠、Ddx3xΔhep 小鼠、Ddx3xfl/fl 小鼠,测定血清 ALT、AST 水平评价肝脏肝损伤情况.结果对F1 代小鼠基因 PCR鉴定的结果表明,基因型符合 Ddx3xfl/fl;F1 代小鼠与 Alb-Cre 小鼠杂交,子代与 Ddx3xfl/fl 小鼠杂交,即获得Ddx3xΔhep 小鼠;PCR与 Western blotting结果显示,目的小鼠肝组织中 Ddx3x基因和蛋白表达显著降低.此外,Ddx3xΔhep 小鼠无胚胎致死现象,出生后生理状况良好.正常饲养条件下,Ddx3xΔhep 小鼠与 Ddx3xfl/fl 小鼠及野生型小鼠相比,血清 ALT、AST 指标及体质量差异均无统计学意义(P>0.05).结论基于 CRISPR/Cas9 联合 Cre-LoxP 方法成功构建了 Ddx3xΔhep 小鼠动物模型,为后续研究Ddx3x在肝脏中的生物学功能及作用机制奠定了良好的基础.

外文标题：Construction and identification of Ddx3x liver conditional knockout mouse model based on CRISPR/Cas9 and Cre-LoxP system

外文摘要：Objective To create a DEAD-box RNA helicase 3X-linked(Ddx3x)liver conditional knockout mouse model by CRISPR/Cas-mediated genome engineering.Methods The Ddx3x-flox mice were constructed by inserting LoxP se-quences into both ends of exons 4-10 of Ddx3x gene by specific sgRNA sequences.F0 and F1 mice were genotyped by PCR.Ddx3x-flox mice were mated with tool mice specifically expressing Alb-Cre to obtain Ddx3x liver conditioned knockout mice(Ddx3xΔhep).PCR and Western blotting were used to detect the expression of target gene and protein in Ddx3xΔhep mice.Ser-um ALT and AST levels were measured in 8-week-old male wild-type C57/6J mice,Ddx3xΔhep mice and Ddx3xfl/fl mice to evaluate liver injury.Results The PCR results of F1 generation mice showed that the genotype was consistent with Ddx3xfl/fl.F1 generation mice were hybridized with Alb-Cre mice,and pups were hybridized with Ddx3xfl/fl mice to obtain Ddx3xΔhep mice.The results of PCR and Western blotting showed that Ddx3x expression decreased significantly in liver tissue of Ddx3xΔhep mice.In addition,Ddx3xΔhep mice did not have embryonal lethality and were in good physiological condition after birth.In normal feeding conditions,Ddx3xΔhep mice had no statistical differences in ALT,AST and body weight from Ddx3xfl/fl mice and wild-type mice.Conclusion The Ddx3xΔhep mice are successfully constructed by CRISPR/Cas-mediated genome engineering,which establish the foundation for the forthcoming research of the biological function and mechanism of Ddx3x in the liver.

外文关键词：

CRISPR/Cas9 technologyCre-LoxP systemDdx3xLiver conditional knockout mice

作者：

潘桢桢、徐玲、张向颖、高耀、田原、范子豪、曹亚玲、任锋

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作者单位：

首都医科大学附属北京佑安医院,北京肝病研究所,北京 100069

关键词：

CRISPR/Cas9技术 Cre-LoxP系统 Ddx3x 肝脏条件性敲除小鼠

基金：

国家自然科学基金项目国家自然科学基金项目北京自然科学基金和北京市教委联合资助重点项目首都卫生发展科研专项重点攻关项目北京市科技计划"首都临床诊疗技术研究及示范应用"专项课题北京市科技计划"首都临床诊疗技术研究及示范应用"专项课题北京市医院管理中心"青苗"计划专项经费资助北京市医管局"登峰"人才计划高层次公共卫生技术人才建设项目

项目编号：

8177061182002243KZ202010025035首发2020-1-1151Z191100006619096Z191100006619097QML20201702DFL20221503学科带头人-02-13

出版年：

2024

DOI：

10.3969/j.issn.1006-5709.2024.10.002

胃肠病学和肝病学杂志

郑州大学

胃肠病学和肝病学杂志

CSTPCD

影响因子：1.029

ISSN：1006-5709

年,卷(期)：2024.33(10)