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首页 > 过刊浏览>2023年第33卷第6期 >35-45,61. DOI:10. 3969 / j.issn.1671-7856. 2023. 06. 005
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Marcksl1 基因敲除对小鼠成年造血的影响
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1.北京协和医学院比较医学中心,中国医学科学院医学实验动物研究所,北京市人类重大疾病实验动物模型工程技术研究中心,国家卫生健康委员会人类疾病比较医学重点实验室,北京 100021;2.北京协和医学院比较医学中心,中国医学科学院医学实验动物研究所,国家人类疾病动物模型资源库,北京 100021

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R-33


Effect of Marcksl1 gene knockout on adult hematopoiesis in mice
Author:
  • GAO Lijuan 2,1

    GAO Lijuan

    1. Comparative Medicine Center, Peking Union Medical College, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Beijing Engineering Research Center for Experimental Animal Models of Human Diseases, Key Laboratory of Human Diseases Comparative Medicine, National Health Commission of China, Beijing 100021, China.
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  • LI Bo 2,1

    LI Bo

    1. Comparative Medicine Center, Peking Union Medical College, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Beijing Engineering Research Center for Experimental Animal Models of Human Diseases, Key Laboratory of Human Diseases Comparative Medicine, National Health Commission of China, Beijing 100021, China.
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  • YU Lei 2,1

    YU Lei

    1. Comparative Medicine Center, Peking Union Medical College, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Beijing Engineering Research Center for Experimental Animal Models of Human Diseases, Key Laboratory of Human Diseases Comparative Medicine, National Health Commission of China, Beijing 100021, China.
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  • QI Xiaolong 2,1

    QI Xiaolong

    1. Comparative Medicine Center, Peking Union Medical College, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Beijing Engineering Research Center for Experimental Animal Models of Human Diseases, Key Laboratory of Human Diseases Comparative Medicine, National Health Commission of China, Beijing 100021, China.
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  • ZHANG Xu 2,1

    ZHANG Xu

    1. Comparative Medicine Center, Peking Union Medical College, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Beijing Engineering Research Center for Experimental Animal Models of Human Diseases, Key Laboratory of Human Diseases Comparative Medicine, National Health Commission of China, Beijing 100021, China.
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  • GAO Shan

    GAO Shan

    2. Comparative Medicine Center, Peking Union Medical College, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, National Human Diseases Animal Model Resource Center, Beijing 100021
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  • LIU Ning

    LIU Ning

    2. Comparative Medicine Center, Peking Union Medical College, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, National Human Diseases Animal Model Resource Center, Beijing 100021
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  • MA Yuanwu

    MA Yuanwu

    1. Comparative Medicine Center, Peking Union Medical College, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Beijing Engineering Research Center for Experimental Animal Models of Human Diseases, Key Laboratory of Human Diseases Comparative Medicine, National Health Commission of China, Beijing 100021, China. 2. Comparative Medicine Center, Peking Union Medical College, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, National Human Diseases Animal Model Resource Center, Beijing 100021
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Affiliation:

1. Comparative Medicine Center, Peking Union Medical College, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Beijing Engineering Research Center for Experimental Animal Models of Human Diseases, Key Laboratory of Human Diseases Comparative Medicine, National Health Commission of China, Beijing 100021, China. 2. Comparative Medicine Center, Peking Union Medical College, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, National Human Diseases Animal Model Resource Center, Beijing 100021

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    摘要:

    目的 建立造血系统特异性Marcksl1 敲除小鼠,探究该基因敲除对小鼠成年造血的影响。 方法对E15. 5 Marcskl1 敲除小鼠进行胎肝竞争性移植实验,分析胎肝移植后不同月龄小鼠外周血中成熟功能谱系血细胞的占比。流式分选胎肝KSL 细胞,利用RNA-Seq 分析该基因敲除后差异基因表达。利用CRISPR/ Cas9 技术,构建Marcksl1 条件敲除小鼠,并与Vav1-Cre 小鼠杂交,获得造血系统特异性Marcksl1 敲除小鼠。利用PCR 和Sanger测序鉴定基因型。利用Real-time PCR 检测小鼠骨髓和造血干细胞中Marcksl1 mRNA 的表达。利用流式细胞技术分析小鼠骨髓、外周血、脾和胸腺中不同类型造血细胞的占比。通过竞争性骨髓移植实验分析Marcksl1 敲除对造血重建的影响。 结果 Marcksl1 敲除造成胎肝移植后B 细胞占比下降、髓系细胞占比升高,骨髓中CLP 和CMP 占比下降、GMP 占比升高。RNA-seq结果显示该基因敲除后导致胎肝KSL 细胞中252 个基因上调,400 个基因下调。GO结果显示差异基因主要富集在免疫应答、质膜以及低压门钙离子通道活性等相关基因。KEGG结果显示差异基因主要富集在造血谱系分化和细胞因子受体互作相关信号通路。我们利用CRISPR/ Cas9 技术成功建立造血特异性Marcksl1 敲除小鼠。流式结果表明Marcksl1 缺失不影响小鼠成年造血,但影响骨髓移植后的造血重建能力。 结论 成功建立造血系统特异性Marcksl1 敲除小鼠。造血系统敲除Marcksl1 基因,不影响成年稳态造血,但影响成年造血重建能力。我们建立的Marcksl1 条件敲除小鼠,为该基因的成年造血功能研究以及该基因的其他组织特异性功能研究提供了动物模型。

    Abstract:

    Objective To establish hematopoietic system-specific Marcksl1 knockout mice and explore the effect of Marcksl1 gene deletion on hematopoiesis. Methods Using fetal liver competitive transplantation of E15. 5 Marcskl1 gene knockout mice, we analyzed the proportions of various hematopoietic cell types in peripheral blood at 1, 2, 3, and 4 months after fetal liver transplantation. KSL cells were sorted by flow cytometry and analyzed by RNA-seq. We used CRISPR/ Cas9 technology to generate Marcksl1 conditional knockout mice and obtained hematopoietic system-specific Marcksl1 knockout mice by crossing with Vav1-Cre mice. The genotype of the produced mice was confirmed by PCR and Sanger sequencing. Real-time PCR was used to assess Marcskl1 mRNA expression in bone marrow and hematopoietic stem cells. Flow cytometry was used to analyze the proportions of various hematopoietic cell types in bone marrow, peripheral blood, spleen, and thymus. The effect of Marcksl1 gene knockout on hematopoietic reconstitution was analyzed by competitive bone marrow transplantation. Results Marcksl1 gene knockout decreased the proportion of B cells, increased the proportion of myeloid cells, decreased CLP and CMP, and increased GMP after fetal liver transplantation. RNA-seq showed that 252 genes were upregulated and 400 genes were downregulated. GO analysis showed that the differentially expressed genes were significantly enriched in the immune response, plasma membrane, and low-pressure gate calcium channel activity. KEGG analysis showed that the differentially expressed genes were significantly enriched in hematopoietic lineage differentiation and cytokine-cytokine receptor interaction-related signaling pathways. We generated hematopoietic system-specific Marcksl1 knockout mice successfully. Flow cytometry showed that Marcksl1 deletion did not affect the steady-state hematopoietic functions of adult mice, but it did affect the hematopoietic reconstitution ability after competitive bone marrow transplantation. Conclusions We successfully established hematopoietic system-specific Marcksl1 knockout mice and found that Marcksl1 gene knockout does not affect stable hematopoiesis, but affects hematopoietic reconstitution. The underlying mechanism requires further investigation. This study provides an animal model to examine the gene function of Marcksl1 in adult hematopoiesis and other aspects.

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高丽娟,李 博,于 磊,齐晓龙,张 旭,高 珊,刘 宁,马元武. Marcksl1 基因敲除对小鼠成年造血的影响[J].中国比较医学杂志,2023,33(6):35~45,61.

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  • 收稿日期:2023-03-15
  • 在线发布日期: 2023-08-18
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