Marcksl1 基因敲除小鼠的建立及造血表型初步分析
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1.中国医学科学院医学实验动物研究所,北京协和医学院比较医学中心,北京市人类重大疾病实验动物模型工程技术研究中心,北京 100021; 2.中国医学科学院医学实验动物研究所,北京协和医学院比较医学中心,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

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R-33

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Establishment of Marcksl1 gene knockout mice and preliminary analysis of a hematopoietic phenotype
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1.Beijing Engineering Research Center for Experimental Animal Models of Human Diseases, Institute of Laboratory Animal Science, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100021, China. 2. Key Laboratory of Human Disease Comparative Medicine, National Health Commission of China (NHC), Institute of Laboratory Animal Science, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100021

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    摘要:

    目的 建立 Marcksl1 基因敲除小鼠,初步探究该基因缺失对造血系统发育的影响。 方法 利用 CRISPR/ Cas9 技术构建 Marcksl1 基因敲除小鼠,通过 PCR 技术以及 Sanger 测序方法鉴定小鼠基因型。 通过将敲除小鼠与野生型小鼠杂交,分析敲除小鼠的传代情况。 通过杂合子小鼠相互杂交,分析发育不同阶段纯合子、杂合子和野生型小鼠的占比。 分离小鼠胎肝,利用血常规以及流式细胞术分析该基因缺失对造血系统的影响。 结果 PCR 结合 Sanger 测序结果表明 Marcksl1 基因敲除小鼠构建成功。对不同阶段胚胎小鼠基因型鉴定和数量统计,发现小鼠 Marcksl1 基因敲除造成的胚胎死亡发生于胚胎发育后期。通过血常规与流式细胞分析,结果表明 Marcksl1 基因缺失在 E15. 5 d 时不影响白细胞,红细胞以及血小板数量,但胎肝中造血干细胞比例显著增多。 结论 本研究成功建立 Marcksl1 基因敲除小鼠,并发现该基因缺失影响造血干细胞占比。本研究为进一步了解该基因在胚胎发育和造血系统中的功能提供动物模型。

    Abstract:

    Objective To generate myristoylated alanine-rich C-kinase substrate-like 1c ( Marcksl1 ) gene knockout mice and investigate the gene function in hematopoiesis. Methods We used CRISPR/ Cas9 technology to produce Marcksl1 gene knockout mice, which was confirmed by PCR and Sanger sequencing. Germline transmission of Marcksl1 gene knockout mice was confirmed by crossing to wild-type mice. We also analyzed the ratio of homozygous, heterozygous, and wild-type mice at different stages of embryonic development. We isolated mice fetal liver to examine the effect of Marcksl1 deletion on the hematopoietic system using routine blood and flow cytometry method . Results PCR and sequencing data showed that Marcksl1 knockout mice were successfully obtained. Our data further suggest that embryonic lethality caused by Marcksl1 gene knockout occurs at a late period of embryonic development. Routine blood and flow cytometry result showed that at E15. 5, deletion of the Marcksl1 gene did not affect the number of white blood cells, red blood cells, or platelets, but the proportion of hematopoietic stem cells in fetal liver increased significantly. Conclusions We have successfully constructed a Marcksl1 knockout mouse. We further found that Marcksl1 deletion increased the number of hematopoietic stem cells. Our work provides a mouse model for further study of the gene function of Marcksl1 in embryonic development and the hematopoietic system.

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李 博,高丽娟,于 磊,张 旭,李争光,刘 宁,史旭东,高 凯,李 静,高 珊,齐晓龙,张连峰,马元武.Marcksl1 基因敲除小鼠的建立及造血表型初步分析[J].中国比较医学杂志,2021,31(5):108~114.

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  • 收稿日期:2020-03-01
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  • 在线发布日期: 2021-06-25
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