基于CRISPR/Cas9系统构建Uox基因敲除的高尿酸血症小鼠模型  

Construction of hyperuricemic mouse model with Uox gene knockout based on CRISPR/Cas9 system

作　　者：张艺薇 龙维虎 唐东红[1] 范胜涛[1] 王鹏 王陈芸 李哲丽[1] 黄璋琼[1] 叶尤松[1] ZHANG Yiwei;LONG Weihu;TANG Donghong;FAN Shengtao;WANG Peng;WANG Chenyun;LI Zheli;HUANG Zhangqiong;YE Yousong(Institute of Medical Biology,Chinese Academy of Medical Sciences&Peking Union Medical College,Kunming 650118,China;Beijing Viewsolid Biotech Co.,Ltd.,Beijing 102200,China)

机构地区：[1]北京协和医学院中国医学科学院医学生物学研究所,昆明650118 [2]北京唯尚立德生物科技有限公司,北京102200

出　　处：《中国实验动物学报》2025年第3期411-419,共9页Acta Laboratorium Animalis Scientia Sinica

基　　金：云南省技术创新人才培养对象项目(202105AD160018);中国医学科学院医学与健康科技创新工程项目(2021-I2M-1-043);国家自然科学基金联合基金项目(U2202214);科技创新2030重大专项(2023ZD0406306)。

摘　　要：目的 通过CRISPR/Cas9技术构建Uox基因敲除且能稳定遗传的小鼠品系,并评价其是否能够模拟高尿酸血症患者的疾病特点。方法 在Uox基因Exon 2~4的前后两侧设计双sgRNA,将基因敲除所需的sgRNA与Cas9 mRNA按照一定比例显微注射进小鼠的受精卵中,培养2~4 h后,将胚胎移植至代孕母鼠体内并最终获得F0代小鼠。对F0代小鼠进行PCR鉴定与测序分析,筛选适合的Uox基因阳性敲除小鼠与野生型(wide type, WT)小鼠合笼获得F1代,再挑选F1代中杂合子(基因型为Uox^(+/-))雌鼠与雄鼠合笼得到纯合的F2代小鼠(基因型为Uox^(-/-))。最后检测Uox^(-/-)小鼠与WT小鼠血清尿酸、尿液尿酸,血清中肌酐、尿素、丙氨酸氨基转移酶、门冬氨酸氨基转移酶的含量并进行对比,通过苏木素-伊红(HE)染色结合Masson染色观察肾和肝组织的病理变化。结果 与WT小鼠相比,Uox^(-/-)小鼠血清尿酸(雄鼠:(478.4±114.6)μmol/L,P<0.001;雌鼠:(507.7±129.6)μmol/L,P<0.001)、尿液尿酸(雄鼠:(4116.8±1928.1)μmol/L,P<0.001;雌鼠:(2998.0±547.7)μmol/L,P<0.01)、血清中肌酐((91.8±55.6)μmol/L,P<0.001)、尿素((28.6±13.9) mmol/L,P<0.05)、丙氨酸氨基转移酶((53.3±23.3) U/L,P<0.01)及门冬氨酸氨基转移酶((203.3±70.3) U/L,P<0.001)水平均显著升高。组织病理学的结果显示,Uox^(-/-)小鼠的肝中可见中量肝细胞变性,肾中可见中重度的肾小管囊性扩张、变性和纤维化,肾小球肥大增生,小血管扩张充血,间质单核及淋巴细胞浸润。结论 通过CRISPR/Cas9技术成功构建了Uox基因Exon 2敲除的小鼠纯合品系,可以作为高尿酸领域相关研究的动物模型。Objective To construct a uricase-deficient mouse model with stable inheritance using the CRISPR/Cas9 system, and evaluate its ability to simulate the disease characteristics of patients with hyperuricemia. Methods Double single guide RNAs(sgRNAs) were designed on both sides of exon 2 ~ 4 of the Uox gene. sgRNA and Cas9 mRNA for gene knockout were microinjected into the fertilized eggs of mice. After culture for 2 ~ 4 h, the embryos were transferred to surrogate mother mice to produce an F0 generation. Uox-knockout mice were identified by polymerase chain reaction and sequencing analysis. Positive mice were then mated with wild-type(WT)mice to produce an F1 generation, and heterozygous female and male F1 mice were then selected to obtain homozygous F2 mice. Serum and urine levels of uric acid, creatinine, and urea, and serum alanine aminotransferase(ALT) and aspartate aminotransferase(AST) levels were detected and compared between homozygous and wild-type mice. Pathological changes in kidney and liver tissues were observed by hematoxylin and eosin and Masson staining. Results Urine levels of serum uric acid(male:(4116.8 ± 1928.1) μmol/L, P < 0.001;female:(2998.0 ± 547.7) μmol/L, P < 0.01) and serum levels of uric acid(male:(478.4 ± 114.6) μmol/L, P < 0.001;female:(507.7 ± 129.6) μmol/L, P < 0.001), creatinine((91.8 ± 55.6) μmol/L, P < 0.001), urea((28.6 ± 13.9) mmol/L, P < 0.05), ALT((53.3 ± 23.3) U/L, P < 0.01), and AST((203.3 ± 70.3) U/L, P < 0.001) were significantly increased in Uox^(-/-) mice compared with WT mice. Histopathological examination showed moderate hepatocyte degeneration in the liver, moderate-to-severe tubular cystic dilation, degeneration, and fibrosis in the kidney, glomerular hypertrophy and hyperplasia, small-vessel dilation and congestion, and infiltration of stromal monocytes and lymphocytes in Uox^(-/-) mice. Conclusions We successfully established a homozygous uricase-deficient mouse strain using CRISPR/Cas9 technology, as a suitable animal model for research in the field of

关 键 词：高尿酸血症 动物模型 CRISPR/Cas9 基因敲除 尿酸氧化酶 

分 类 号：Q95-33[生物学—动物学]