小鼠Brd2基因探针的制备及其在荧光原位杂交实验中的应用

The preparation of Brd2 gene probes of mouse and their application in the fluorescent in situ hybridization experiment

目的:为观察溴结构域包含蛋白2(bromodomain containing protein 2,Brd2)基因在小鼠中枢神经系统的表达与分布,本研究制备了两条地高辛标记的Brd2 cRNA探针。方法:提取小鼠脑组织总RNA,设计两对Brd2引物,通过逆转录聚合酶链式反应(RT-PCR)方法,扩增得到两个Brd2的DNA片段,并将它们分别克隆到pCRⅡ-TOPO载体中。利用体外转录方法合成地高辛标记的cRNA探针,最后通过荧光原位杂交实验分析所标记探针的特异性及杂交效果。结果:本实验成功构建了两个Brd2/pCRⅡ-TOPO质粒,获得了特异性的地高辛标记的Brd2cRNA探针,在荧光原位杂交实验中应用两条探针得到了较好的杂交信号。结论:本实验制备的地高辛标记的cRNA探针可特异地检测Brd2 mRNA,为进一步观察Brd2 mRNA在中枢神经系统的分布及功能研究提供了工具。

Objective In order to observe bromodomain containing protein 2 （Brd2） gene expression in the centralnervous system of the mouse, we prepared two digoxigenin （DIG）- labeled Brd2 cRNA probes. Methods： Total RNAwas extracted from mouse brain and two pairs of Brd2 gene primers were designed. Then the two Brd2 DNA fragmentswere obtained through RT-PCR method and they were cloned into the pCR II -TOPO vectors respectively. Using in vitrotranscription method, two DIG-labeled probes were generated. The specificity of the two probes was detected by using flu-orescent in situ hybridization. Results： We successfully constructed two Brd2/pCR II-TOPO plasmids, and obtained twoDIG-labeled Brd2 cRNA probes. Fluorescent in situ hybridization results showed good hybridization signals. Conclusion：The cRNA probes of Brd2 for fluorescent in situ hybridization were prepared in this study, which will provide an approachto further study on the expression and function of Brd2 in the central nervous system.