上海交通大学学报（医学版）

›› 2013, Vol. 33 ›› Issue (3): 257-.doi: 10.3969/j.issn.1674-8115.2013.03.001

• 论著（基础研究） •    下一篇

转Notch1基因T淋巴细胞白血病双荧光示踪斑马鱼模型的建立及鉴定

卿 恺1, 陈芳源1, 沈莉菁1, 林文洁1, 孙瑞林2, 钟济华1   

  1. 1.上海交通大学 医学院附属仁济医院血液科, 上海 200127； 2.上海南方模式生物研究中心, 上海 201203
  • 出版日期:2013-03-28 发布日期:2013-03-29
  • 通讯作者: 陈芳源, 电子信箱: chenfy04@yahoo.com.cn。
  • 作者简介:卿 恺（1986—）, 男, 硕士生； 电子信箱: qingkai_xx@163.com。
  • 基金资助:

    上海市卫生局重大课题基金（ZYSNXD-CC-ZDYJ001）；上海市科委中医引导项目基金（12401906700）；上海交通大学医学院附属仁济医院科研种子基金(RJZZ12-007)

Construction and identification of Notch1 gene induced T-cell leukemia with double florescence tracer in transgenic zebrafish

QING Kai1, CHEN Fang-yuan1, SHEN Li-jing1, LIN Wen-jie1, SUN Rui-lin2, ZHONG Ji-hua1   

  1. 1. Department of Hematology, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200127, China； 2.Shanghai Research Center for Biomodel Organisms, Shanghai 201203, China
  • Online:2013-03-28 Published:2013-03-29
  • Supported by:

    Shanghai Municipal Health Bureau Foundation, ZYSNXD-CC-ZDYJ001； Shanghai Science and Technology Committee Foundation, 12401906700； Foundation of Renji Hospital, Shanghai Jiaotong University School of Medicine, RJZZ12-007

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摘要：

目的 在血管内皮增强型绿色荧光蛋白标记的斑马鱼系（fli1-EGFP）中，转入Rag2启动子驱动的人源性截短型Notch1基因（ICN1），从中筛选并鉴定发生急性T淋巴细胞白血病(T-ALL)的斑马鱼，建立稳定传代的红绿双色示踪的T-ALL斑马鱼疾病模型。方法 构建Rag2-ICN1-EGFP质粒，显微注射到斑马鱼胚胎的单细胞期，利用荧光体视显微镜筛选F0代，并建立稳定传代的转基因鱼系。通过荧光体视显微镜、RT-PCR、Western blotting验证ICN1 mRNA和蛋白的表达，并利用流式细胞术、外周血涂片方法鉴定T-ALL斑马鱼模型的表型。结果 在867条显微注射重组质粒的斑马鱼中，鉴定发现有20条（2.3%）转基因阳性斑马鱼，为F0代转基因斑马鱼，其中雄鱼9条，雌鱼11条。在已性成熟的F0 代成鱼之间采取一对一形式交配，产下F1代，通过荧光体视显微镜、RT-PCR、Western blotting在大体观、mRNA以及蛋白水平验证了ICN1的表达；流式细胞术和外周血涂片结果显示，转基因F1代斑马鱼与fli1EGFP斑马鱼相比,红细胞比例显著降低，淋巴细胞比例明显增高。结论 成功构建转Notch1基因红绿双色示踪的T-ALL斑马鱼模型，为深入探讨Notch在促进TALL发生中的调控机制以及利用这种疾病模型进行高通量的活体药物筛选提供了研究平台。

关键词: Notch1, 急性T淋巴细胞白血病, 斑马鱼, 双荧光示踪

Abstract:

Objective To establish stable germline ICN1 （intracellular domain Notch1）transgenic and double tracer T acute lymphoid leckemia, T-ALL zebrafish models by overexpressing human ICN1 gene in fli-EGFP zebrafish through T lymphocytes specific Rag2 promoter. Methods ICN1 plasmid carrying red fluorescent protein “reporter gene” (Rag2-ICN1-EGFP) was constructed and microinjected into zebrafish single-cell embryos, then the F0 (founder) generation was identified by fluorescence screening for establishing the zebrafish stable transgenic germline. The expression of ICN1 gene and protein was determined by fluorescence microscope, RT-PCR and Western blotting, and the phenotype of T-ALL zebrafish models was identified by flow cytometry and peripheral blood smear. Results Twenty of the 867 (2.3%) mosaic F0 zebrafish embryos injected with the constructed vector were identified to be the germline transgenic zebrafish, including 9 males and 11 females. The F0 adult fish of sexual maturity was copulated as one-to-one form, then F1 generation was produced. By using general view fluorescence microscope, RT-PCR and Western blotting, the expression of ICN1 was validated at the general view, mRNA and protein levels. Flow cytometry and peripheral blood smears suggested a significant reduction in the proportion of red blood cells and an increase of lymphocyte ratio in transgenic zebrafish as compared to control group. Conclusion Notch1 gene induced T-ALL zebrafish models with double tracer system have been successfully constructed, which may provide a research platform for the in-depth discussion on the ICN1 in the promotion of regulatory mechanism of T-ALL and high throughput drug screening in vivo.

Key words: Notch1, T acute lymphoid leukemia, zebrafish, double florescence tracer