庹媛媛;尚鲁俊;夏海雄;何志旭;舒莉萍;
TUO Yuanyuan;SHANG Lujun;XIA Haixiong;HE Zhixu;SHU Liping;Department of Pediatrics,the Affiliated Hospital of Guizhou Medcial University;Department of Immunology,Guizhou Medical University;Laboratory Animal Center,Guizhou Medical University;Tissue Engineering and Stem Cell Research Center,Guizhou Medcial University;

• 1:贵州医科大学儿科教研室
• 2:贵州医科大学免疫学教研室
• 3:贵州医科大学实验动物中心
• 4:贵州医科大学组织工程与干细胞实验中心

摘要(Abstract)：

目的:用band3基因的反义mRNA探针进行斑马鱼全胚胎原位杂交,检测band3基因在野生型Tüebingen斑马鱼胚胎发育过程中的表达。方法:Trizol法提取斑马鱼胚胎总RNA并设计特异性引物,采用RTPCR法扩增band3基因cDNA编码区片段;通过基因重组技术构建PBSK-band3重组质粒,用Bam HⅠ酶切得到线性化PBSK-band3;以T7 RNA聚合酶转录合成反义地高辛标记的band3 RNA探针,用全胚胎原位杂交法检测band3基因在斑马鱼胚胎早期的表达情况。结果:成功克隆band3基因片段,构建PBSK-band3重组质粒;经体外转录获得地高辛标记的反义band3 mRNA探针,原位杂交后检测证实band3基因在斑马鱼胚胎早期发育过程中间细胞群(ICM)和主动脉性腺中肾区(AGM)呈现高表达。结论:地高辛标记的反义band3 mRNA探针可检测到斑马鱼胚胎中band3基因的定位表达。
Objective: The antisense mRNA probe of band3 gene was adopted to conduct Zebrafish whole embryos mount in situ hybridization to detect the expression of band3 gene in wild-type Tebingen zebrafish embryonic development. Method: Zebrafish embryo total RNA was extracted by Trizol,specific primers was designed and the c DNA coding region fragment band3 was amplified by RT-PCR. The recombinant PBSK / band3 plasmids was constructed by recombination technique and linearized with Bam H I enzyme digestion. T7 RNA polymerase was adopted to synthesize antisense band3 RNA probes labeled by digoxigenin. Zebrafish whole embryos mount in situ hybridization was adopted to detect band3 gene expression in early zebrafish embryos. Results: The band3 gene fragments were cloned and PBSK / band3 recombinant plasmid was constructed successfully. The digoxigenin-labeled antisense band3 probe was obtained in vitro transcription. After mount in situ hybridization,it was observed that band3 gene was highly expressed in the middle cell population( ICM) and the renal region( AGM) of the aorta gonad in the early embryonic development of zebrafish. Conclusion: In this study,we prepare the antisense mRNA band3 probe labeled with digoxigenin and detect the expression of band3 gene in zebrafish embryos.

关键词(KeyWords)： 斑马鱼;胚胎;band3;全胚胎原位杂交;探针
zebrafish;embryo;band3;whole embryo mount in situ hybridization;probe

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(31260284;31360285);; 贵州省科技基础条件平台项目[(2009)4005];; 贵州省优秀科技省长专项基金(S2008-4)

作者(Author): 庹媛媛;尚鲁俊;夏海雄;何志旭;舒莉萍;
TUO Yuanyuan;SHANG Lujun;XIA Haixiong;HE Zhixu;SHU Liping;Department of Pediatrics,the Affiliated Hospital of Guizhou Medcial University;Department of Immunology,Guizhou Medical University;Laboratory Animal Center,Guizhou Medical University;Tissue Engineering and Stem Cell Research Center,Guizhou Medcial University;

Email:

DOI: 10.19367/j.cnki.1000-2707.2016.07.004

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