[地高辛标记的斑马鱼cd99l2基因RNA探针的制备]
[Preparation of RNA probe for cd99l2 gene of zebrafish labeled with digoxingenin-UTP].
作者信息
Wen Zong-hua, Zhang Yan, Wu Zi-qin, Zhou Xin-hua, Han Xi-qun, Zhang Wen-qing, Zhao Tong
机构信息
Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
出版信息
Nan Fang Yi Ke Da Xue Xue Bao. 2010 May;30(5):969-72.
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 T(7) 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.
目的
为研究cd99l2基因在斑马鱼发育过程中的表达模式，本研究制备了用于整体原位杂交的RNA探针。
方法
将通过RT-PCR获得的cd99l2片段克隆到pGM-T Easy中，然后用限制性内切酶SacII或SalI将质粒线性化。使用Sp6或T(7) RNA聚合酶合成地高辛标记的反义探针和正义探针，并通过整体原位杂交进行验证。
结果
构建了质粒cd99l2/pGM-T。使用反义探针检测了斑马鱼胚胎发育过程中cd99l2基因的表达模式，在斑马鱼发育过程中，中枢神经系统中检测到强烈表达。
结论
以正义探针为对照，反义探针可用于研究cd99l2在斑马鱼发育过程中的时空分布。
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