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Generation of Brown Fat-Specific Knockout Mice Using a Combined Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus Single-Guide RNA System
Published on: March 24, 2023
[Generation and phenotypic characterization of S100A9 gene knockout mice by CRISPR/Cas9-mediated gene targeting]
Pei Yan1,2, Da-Yan Liang1,2, Wen-Hao Xu1,2
1Institute of Medical Biology, College of Life Sciences, 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.
Insights
Researchers successfully created S100A9 gene-edited mice using CRISPR/Cas9 technology. These S100A9 knockout mice showed aggravated airway inflammation, highlighting S100A9
Area of Science:
- Genetics and Genomics
- Immunology
- Molecular Biology
Background:
- S100 calcium binding protein A9 (S100A9) plays a role in inflammation and tumor progression.
- Understanding S100A9's function requires appropriate animal models.
Purpose of the Study:
- To generate S100A9 gene-edited mice utilizing CRISPR/Cas9 technology.
- To establish a novel animal model for investigating S100A9's biological roles.
Main Methods:
- CRISPR/Cas9 microinjection into mouse fertilized eggs to target S100A9 exons 2 and 3.
- Generation and identification of S100A9 knockout (S100A9-/-) mice through PCR and sequencing.
- Verification of gene knockout and protein expression using real-time PCR, Western blot, and immunohistochemistry.
- Induction of allergic asthma model using ovalbumin (OVA) for pathological analysis via HE staining and bronchoalveolar lavage fluid (BALF) examination.
Main Results:
- Successful knockout of the S100A9 gene (2,492 bp deletion in exons 2-3) and generation of stably inherited S100A9-/- mice.
- High S100A9 expression observed in the lungs and spleen of wild-type mice; absence of S100A9 mRNA and protein in S100A9-/- mice.
- S100A9-/- mice exhibited a significantly exacerbated inflammatory lung phenotype and increased eosinophils in BALF upon OVA challenge compared to wild-type controls.
Conclusions:
- Successfully constructed a new strain of S100A9 knockout mice.
- Preliminary findings indicate that S100A9 deficiency exacerbates airway inflammation in an allergic asthma model.
- This S100A9-/- mouse model provides a valuable tool for further research into S100A9's functions in biological processes.
