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  

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

作　　者：潘桢桢 徐玲 张向颖[1] 高耀 田原 范子豪 曹亚玲 任锋[1] PAN Zhenzhen;XU Ling;ZHANG Xiangying;GAO Yao;TIAN Yuan;FAN Zihao;CAO Yaling;REN Feng(Beijing Institute of Hepatology,Beijing Youan Hospital,Capital Medical University,Beijing 100069,China)

机构地区：[1]首都医科大学附属北京佑安医院,北京肝病研究所,北京100069

出　　处：《胃肠病学和肝病学杂志》2024年第10期1275-1280,共6页Chinese Journal of Gastroenterology and Hepatology

基　　金：国家自然科学基金项目(81770611、82002243);北京自然科学基金和北京市教委联合资助重点项目(KZ202010025035);首都卫生发展科研专项重点攻关项目(首发2020-1-1151);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(Z191100006619096、Z191100006619097);北京市医院管理中心“青苗”计划专项经费资助(QML20201702);北京市医管局“登峰”人才计划(DFL20221503);高层次公共卫生技术人才建设项目(学科带头人-02-13)。

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(Ddx3x^(Δhep))。采用PCR方法和Western blotting方法分别检测Ddx3x^(Δhep)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、Ddx3x^(Δhep)小鼠、Ddx3x^(fl/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Ddx3x^(fl/fl);F1代小鼠与Alb-Cre小鼠杂交,子代与Ddx3x^(fl/fl)小鼠杂交,即获得Ddx3x^(Δhep)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,Ddx3x^(Δhep)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,Ddx3x^(Δhep)小鼠与Ddx3x^(fl/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Ddx3x^(Δhep)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。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 sequences 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(Ddx3x^(Δhep)).PCR and Western blotting were used to detect the expression of target gene and protein in Ddx3x^(Δhep) mice.Serum ALT and AST levels were measured in 8-week-old male wild-type C57/6J mice,Ddx3x^(Δhep) mice and Ddx3x^(fl/fl) mice to evaluate liver injury.Results The PCR results of F1 generation mice showed that the genotype was consistent with Ddx3x^(fl/fl).F1 generation mice were hybridized with Alb-Cre mice,and pups were hybridized with Ddx3x^(fl/fl) mice to obtain Ddx3x^(Δhep) mice.The results of PCR and Western blotting showed that Ddx3x expression decreased significantly in liver tissue of Ddx3x^(Δhep) mice.In addition,Ddx3x^(Δhep) mice did not have embryonal lethality and were in good physiological condition after birth.In normal feeding conditions,Ddx3x^(Δhep) mice had no statistical differences in ALT,AST and body weight from Ddx3x^(fl/fl) mice and wild-type mice.Conclusion The Ddx3x^(Δhep) 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.

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

分 类 号：R575[医药卫生—消化系统]