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Generation and phenotypic characterization of S100A9 gene knockout mice by CRISPR/Cas9-mediated gene targeting

YAN Pei^1,2, LIANG Da-Yan^1,2, XU Wen-Hao^1,2, XUE Lu^1,2, YU Meng-Fei^1,2, SHEN Jin-Hua^1,2, LIU Qing-Hua^1,2, PENG Yong-Bo^1,2,*

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

Abstract

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.

Key words: CRISPR/Cas9; S100A9; gene editing; mouse

Received: 2020-09-30　　Accepted: 2021-02-08

Corresponding author: 彭勇波　　E-mail: pyb1980@mail.scuec.edu.cn

DOI: 10.13294/j.aps.2021.0045

Citing This Article：

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).