地高辛标记斑马鱼cd99l2基因RNA探针的制备  

Preparation of RNA probe for cd99l2 gene of zebrafish labeled with digoxingenin-UTP

作　　者：温宗华[1] 张艳[2] 吴自勍[2] 周新华[2] 韩西群[2] 张文清[3] 赵彤[1,2] 

机构地区：[1]南方医科大学南方医院病理科,广东广州510515 [2]南方医科大学广东省分子肿瘤病理学重点实验室,广东广州510515 [3]南方医科大学细胞生物学教研室,广东广州510515

出　　处：《南方医科大学学报》2010年第5期969-972,共4页Journal of Southern Medical University

基　　金：国家自然科学基金(30770908)

摘　　要：目的制备用于斑马鱼胚胎早期cd99l2基因时空表达检测的地高辛标记RNA探针。方法设计引物,构建cd99l2/pGM-T重组质粒,用SacII和SalI分别进行酶切得到线性化DNA片段,以Sp6RNA聚合酶和T7RNA聚合酶转录合成地反义和正义地高辛标记的RNA探针,用整体原位杂交法检测斑马鱼胚胎早期cd99l2基因的表达。结果成功构建了cd99l2/pGM-T重组质粒,体外转录获得反义和正义RNA探针,反义探针杂交检测证实cd99l2基因在斑马鱼胚胎早期发育过程中中枢神经系统呈现高表达,正义探针未检测到表达信号。结论本研究制备的反义cd99l2RNA探针,兼顾了特异性和敏感性,能有效检测斑马鱼胚胎早期cd99l2基因的定位表达,而正义探针可以作为阴性对照,为进一步探究cd99l2基因在斑马鱼发育过程中的作用机制奠定了基础。Objective To study the expression pattern of cd99l2 gene during zebrafish development, the RNA probes for whole-mount in situ hybridization were prepared in this study. Methods The cd99l2 fragment obtained by RT-PCR was cloned into pGM-T Easy, then the plasmids were linearized with the restriction enzymes SacII or SalI. Using Sp6 or T7 RNA polymerase, the digoxingenin-labeled antisense and sense probes were synthesized and confirmed by whole-mount in situ hybridization. Results The plasmid cd99l2/pGM-T was constructed. cd99l2 gene expression pattern during embryogenesis of zebrafish was examined using the antisense probe, and intense expression was detected in the central nervous system during zebrafish development. Conclusion The antisense probe can be used for study of the spatial and temporal distribution of cd99l2 during zebrafish development using the sense probe as control.

关 键 词：斑马鱼 原位杂交 探针 cd99l2 

分 类 号：Q78[生物学—分子生物学]