摘要
目的:探讨cdh 5-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cdh 5基因片段,将cdh 5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+-cdh 5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cdh 5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cdh 5-mRNA杂交信号并拍照。结果:构建并鉴定了pCS^2+-cdh 5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cdh 5-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cdh 5-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 cdh 5 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+-cdh 5 recombinant plasmid was verified by double enzyme digestion and sequencing.Digoxin labeled antisense cdh5 mRNA was obtained by using linearized pCS^2+-cdh 5 as a template by T3RNA transcription system,and served as a probe for whole-mount in situ hybridization of zebrafish embryos.The cdh 5-mRNA hybridization signal of embryos at all developmental phases were observed under microscope and photographed.Results:The recombinant plasmid pCS^2+-cdh 5 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.
作者
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(Department of Tissue Engineering and Stem Cell Experiment,Guizhou Medical University,Guiyang 550004,Guizhou,China;Department of Immunology,Guizhou Medical University,Guiyang 550004,Guizhou,China;Department of Pediatrics,Hebei Provincial People's Hospital,Shijiazhuang 050057,Hebei,China;Department of Critical Care Medicine,The Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China;Department of Childhood Oncology,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China)
出处
《贵州医科大学学报》
CAS
2019年第11期1241-1246,共6页
Journal of Guizhou Medical University
基金
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]
关键词
斑马鱼
胚胎发育
CDH
5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
