严珮1,2, 梁大焱1,2, 许文豪1,2, 薛璐1,2, 于孟飞1,2, 沈金花1,2, 刘庆华1,2, 彭勇波1,2,*
1中南民族大学生命科学学院医学生物研究所，武汉 430072；2中南民族大学武陵山区特色资源植物保护与利用湖北省重点实验室，武汉 430072
S100钙结合蛋白A9 (S100 calcium binding protein A9, S100A9)参与多种炎症反应及肿瘤细胞的迁移和侵袭调控等生物学过程。本研究目的是利用CRISPR/Cas9技术构建S100A9基因编辑小鼠，为探讨该基因生物学功能提供动物模型。根据S100A9基因序列构建针对外显子2和3的单链小向导RNA (small guide RNA, sgRNA)，体外转录后，通过显微注射将Cas9 mRNA和候选sgRNA混合物注射到小鼠受精卵中，体外培养获得早期胚胎并移植到代孕小鼠，所获F0小鼠经PCR鉴定和基因测序鉴定其基因型。F0小鼠进一步与野生型C57BL/6小鼠测交获得F1杂合子小鼠，再通过F1小鼠自交获得纯合子后代，并利用real-time PCR、Western blot及免疫组织化学(immunohistochemistry, IHC)等方法验证S100A9表达和分布情况；构建鸡卵清白蛋白(ovalbumin from chicken egg white, OVA)激发的过敏性哮喘模型，HE染色观察小鼠肺组织病理变化。结果显示，本方法成功敲除S100A9基因2、3外显子2 492 bp，并获得能稳定遗传的S100A9−/−小鼠。S100A9基因在野生型小鼠的肺和脾脏中高表达，而在S100A9−/−小鼠肺和脾脏中未检测到S100A9 mRNA和蛋白的表达；在表型方面，S100A9−/−小鼠在生长、繁殖及发育方面未表现出明显的缺陷，但在OVA处理条件下，相较于野生型小鼠，S100A9−/−小鼠肺部表现出明显加重的炎症表型，且支气管肺泡灌洗液(bronchoalveolar lavage fluid, BALF)中嗜酸性粒细胞的占比显著升高。以上结果表明，本研究成功构建了S100A9基因编辑小鼠新品系，并初步证实S100A9功能缺失加重哮喘小鼠气道炎症，为研究S100A9基因功能提供了新的小鼠模型。
Generation and phenotypic characterization of S100A9 gene knockout mice by CRISPR/Cas9-mediated gene targeting
YAN Pei1,2, LIANG Da-Yan1,2, XU Wen-Hao1,2, XUE Lu1,2, YU Meng-Fei1,2, SHEN Jin-Hua1,2, LIU Qing-Hua1,2, PENG Yong-Bo1,2,*
1Institute of Medical Biology, College of Life Sciences, South-Central University for Nationalities, Wuhan 430072, China ；2Hubei Provincial Key Laboratory of Plant Protection and Utilization of Characteristic Resources in Wuling Mountain, South-Central University for Nationalities, Wuhan 430072, China
S100 calcium binding protein A9 (S100A9) is involved in a variety of biological processes such as inflammation and tumor cell migration and invasion regulation. The purpose of this study was to construct S100A9 gene-edited mice by using CRISPR/Cas9 technology, thereby providing an animal model for exploring the biological functions of this gene. According to the S100A9 gene sequence, the single-stranded small guide RNA (sgRNA) targeting exons 2 and 3 was transcribed in vitro, and a mixture of Cas9 mRNA and candidate sgRNA was injected into mouse fertilized eggs by microinjection. Early embryos were obtained and transferred to surrogate mice, and F0 mice were obtained and identified by PCR identification and gene sequencing. F0 mice were further mated with wild-type C57BL/6 mice to obtain F1 heterozygous mice, and then homozygous offspring were obtained through F1 mice self-crossing. Real-time PCR, Western blot and immunohistochemistry (IHC) were used to verify the expression and distribution of S100A9. In order to observe the pathological changes of mouse lung tissue using HE staining, an allergic asthma model was induced by ovalbumin from chicken egg white (OVA). The results showed that the 2 492 bp of exons 2, 3 of the S100A9 gene was successfully knocked out, and S100A9−/− mice with stable inheritance were obtained. Furthermore, it was found that S100A9 gene was highly expressed in the lung and spleen of wild-type mice. The expression of S100A9 mRNA and protein was not detected in the lung and spleen of S100A9−/− mice. However, compared with wild-type mice, the lungs of S100A9−/− mice showed a significantly worse inflammatory phenotype, and the proportion of eosinophils in bronchoalveolar lavage fluid (BALF) was significantly increased in response to the treatment of OVA. These results suggest we have successfully constructed a new strain of S100A9−/− mice, and preliminarily confirmed that the lack of S100A9 function can aggravate airway inflammation in asthmatic mice, providing a new mouse model for further study of S100A9 gene function.
通讯作者：彭勇波 E-mail: firstname.lastname@example.org
严珮, 梁大焱, 许文豪, 薛璐, 于孟飞, 沈金花, 刘庆华, 彭勇波. CRISPR/Cas9技术制备S100A9基因编辑小鼠及表型研究[J]. 生理学报 2021; 73 (3): 482-490.
YAN Pei, LIANG Da-Yan, XU Wen-Hao, XUE Lu, YU Meng-Fei, SHEN Jin-Hua, LIU Qing-Hua, PENG Yong-Bo. Generation and phenotypic characterization of S100A9 gene knockout mice by CRISPR/Cas9-mediated gene targeting. Acta Physiol Sin 2021; 73 (3): 482-490 (in Chinese with English abstract).