吴西军,姚冬静,刘庆,杨小燕,王念雪,王一慧,舒莉萍,何志旭
WU Xijun,YAO Dongjing,LIU Qing,YANG Xiaoyan,WANG Nianxue,WANG Yihui,SHU Liping,HE Zhixu

• 1:贵州医科大学组织工程与干细胞实验中心
• 2:贵州医科大学免疫学教研室
• 3:河北省人民医院儿科
• 4:贵州医科大学附院呼吸与危重症医学科

摘要(Abstract)：

目的:探讨cdh5-mRNA在野生型斑马鱼胚胎期发育过程中的表达模式。方法:收取tuebingen野生型斑马鱼多个发育时间节点的胚胎,提取其总RNA,体外逆转录后RT-PCR扩增cdh5基因片段,将cdh5基因片段插入pCS~(2+)质粒载体中,挑选阳性单克隆菌落,通过双酶切、测序等方法鉴定pCS~(2+)-cdh5重组质粒;重组质粒单酶切后用T3RNA体外转录体系制备地高辛标记的cdh5基因的反义mRNA探针,对斑马鱼全胚胎进行原位杂交,显微镜下观察各发育节点胚胎cdh5-mRNA杂交信号并拍照。结果:构建并鉴定了pCS~(2+)-cdh5重组质粒,各发育节点胚胎经全胚胎原位杂交后发现在tuebingen野生型斑马鱼胚胎发育至3.7～12 hpf时cdh5-mRNA呈低丰度泛在表达,18～36 hpf时在后脑及血管处表达、在造血系统高表达,48 hpf后仅在脑部和血管处低表达。结论:在野生型斑马鱼胚胎期发育过程中可以检测到cdh5-mRNA在后脑和血管处有表达,并且在造血组织高表达。
Objective: To investigate the expression pattern of cdh5 gene during embryogenesis of wild-type zebrafish.Methods: Total RNA was extracted from the embryos with several developmental phases of Tuebingen wild type zebrafish, and the cdh5 gene fragment was amplified by RT-PCR after reverse transcription. The cdh5 gene fragment was ligated into the pCS~(2+) plasmid vector. The pCS~(2+)-cdh5 recombinant plasmid was verified by double enzyme digestion and sequencing. Digoxin labeled antisense cdh5 mRNA was obtained by using linearized pCS~(2+)-cdh5 as a template by T3 RNA transcription system, and served as a probe for whole-mount in situ hybridization of zebrafish embryos. The cdh5-mRNA hybridization signal of embryos at all developmental phases were observed under microscope and photographed. Results: The recombinant plasmid pCS~(2+)-cdh5 was constructed and verified. In situ hybridization showed that the cdh5-mRNA was lowly but ubiquitously expressed at 3.7～12 hpf, highly expressed in the hematopoietic system at 18～36 hpf, highly expressed in tritocerebrum and blood vessels, while only low expressed in tritocerebrum and blood vessels after 48 hpf. Conclusion: During the embryonic development of wild zebrafish, cdh5-mRNA can be detected in tritocerebrum vessels, and was highly expressed in hematopoietic tissues.

关键词(KeyWords)： 斑马鱼;胚胎发育;cdh5基因;基因表达;遗传载体;全胚胎原位杂交
zebrafish;embryonic development;cdh5 gene;gene expression;genetic vector;whole-mount in situ hybridization

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金地区项目(81760025);; 中国医学科学院中央级公益性科研院所基本科研业务费专项资金(2017PT31042,2018PT31048);; 贵阳市科技计划项目(筑科合同201500103);; 贵州省教育厅青年成长基金[黔教合(2018)192]

作者(Author): 吴西军,姚冬静,刘庆,杨小燕,王念雪,王一慧,舒莉萍,何志旭
WU Xijun,YAO Dongjing,LIU Qing,YANG Xiaoyan,WANG Nianxue,WANG Yihui,SHU Liping,HE Zhixu

DOI: 10.19367/j.cnki.1000-2707.2019.11.001

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