2020 年第 7 期 第 15 卷

CRISPR/Cas9基因编辑技术构建5型磷酸二酯酶基因敲除鼠的模型及表型分析

Construction of phosphodiesterase 5 knockout mouse model by CRISPR/Cas9 and its phenotype analysis

作者：马友才李思懿聂绍平公威

英文作者：Ma Youcai Li Siyi Nie Shaoping Gong Wei

单位：首都医科大学附属北京安贞医院急诊危重症中心北京市心肺血管疾病研究所100029

英文单位：Department of Emergency Critical Care Center Beijing Anzhen Hospital Capital Medical University Beijing Institute of Heart Lung and Blood Vessel Diseases Beijing 100029 China

关键词：CRISPR/Cas9基因编辑；5型磷酸二酯酶；基因敲除；表型

英文关键词：CRISPR/Cas9；Phosphodiesterase5；Knockout；Phenotype

• 摘要：

• 目的利用CRISPR/Cas9基因编辑技术建立5型磷酸二酯酶（PDE5）基因敲除小鼠模型并分析小鼠表型，初步探讨PDE5基因敲除对小鼠各项生命指标的影响。方法利用CRISPR/Cas9基因编辑技术,通过胚胎显微注射移植法引导Cas9裂解酶裂解PDE5基因，删除第2外显子589个核苷酸构建敲除小鼠模型，鼠尾DNA做聚合酶链反应和测序鉴定基因型，将阳性PDE5基因敲除鼠和C57BL/6J背景鼠回交，再次通过鼠尾DNA做聚合酶链反应和测序鉴定基因型验证阳性PDE5敲除鼠并繁育。选择8只雄性C57BL/6J野生型小鼠及8只雄性PDE5基因敲除纯合子小鼠，通过观察2组小鼠生长发育情况，利用病理切片和超声心动图检测心脏结构和功能，检测全血细胞计数和生化指标，进行开场实验，分析PDE5基因敲除小鼠的表型。结果PDE5基因敲除小鼠已成功繁育鉴定，子代基因敲除纯合型小鼠无胚胎期致死现象且生长正常，与野生型小鼠相比外观、生长发育未见明显差异。PDE5基因敲除对小鼠的心脏形态及功能无明显影响；但基因敲除鼠的血小板计数、中性粒细胞计数及百分比均明显低于野生鼠［(894±74)×109/L比(1 052±150)×109/L、(1.3±0.4)×109/L比(1.8±0.4)×109/L、(14±3)%比(18±4)%］（均P＜0.05）。同时，基因敲除鼠开场实验中0～5 min和5～10 min的全场运动距离和全场运动速度明显高于野生鼠［全场运动距离：（18 345±5 480）mm比（12 057±3 154）mm、（15 249±3 581）mm比（10 190±1 869）mm；全场运动速度：（61±18）mm/s比（40±11）mm/s、（51±12）mm/s比（34±6）mm/s］（均P＜0.05）。结论经基因型鉴定已成功获得 PDE5基因敲除小鼠。表型分析提示，PDE5基因敲除鼠较野生鼠而言，血小板和中性粒细胞水平降低，活动力明显增强，但PDE5基因敲除对小鼠心脏形态及功能未见明显影响。

• ObjectivePhosphodiesterase 5 (PDE5) knockout mouse model was established by CRISPR/Cas9 technology, and the phenotype was analyzed to preliminarily explore the effect of PDE5 knockout on various life indicators of mice. Methods Embryo microinjection method and deleting exon2 (589 bp) wers used to create PDE5 knockout mouse model by CRISPR/Cas9-mediated genome engineering. The knockout mice were genotyped by polymerase chain reaction (PCR), followed by sequence analysis. The positive PDE5 knockout mice were backcrossed with C57BL/6J mice, and the pups were genotyped by PCR, followed by sequence analysis, and positive PDE5 knockout mice began to breed. Eight male C57BL/6J wild mice and 8 male PDE5 knockout homozygous mice were chosen, the growth and development of the two groups of mice were observed. Pathological sections and echocardiography were used to detect the structure and function of the heart. The whole blood cell count, biochemical examination, and the opening experiment were used to analyze the phenotype of the PDE5 knockout mice. Results  PDE5 knockout mice have been successfully bred and identified, and the homozygous knockout mice had no death in embryo and grew normally, with no significant difference in appearance, growth and development compared with wild mice. PDE5 knockout had no significant effect on cardiac morphology and function. However, the platelet count, neutrophil count and neutrophil percentage in knockout mice were significantly lower than those in wild mice ［(894±74)×109/L vs (1 052±150)×109/L,(1.3±0.4)×109/L vs (1.8±0.4)×109/L,(14±3)% vs (18±4)%］(all P＜0.05). At the same time, the whole area distance and whole area velocity in the first 0-5 min and 5-10 min of PDE5 knockout mice were significantly higher than those in the wild mice ［whole area distance：（18 345±5 480）mm vs （12 057±3 154）mm,（15 249±3 581）mm vs （10 190±1 869）mm； whole area velocity：（61±18）mm/s vs （40±11）mm/s,（51±12）mm/s vs （34±6）mm/s］(all P＜0.05). Conclusions  PDE5 gene knockout mice have been successfully obtained by genotype identification. Phenotypic analysis indicated that compared with wild mice, the levels of platelets and neutrophils were decreased and the activity was significantly increased in PDE5 knockout mice. PDE5 gene knockout has no obvious effect on mouse heart morphology and function.