基于MS2衣壳蛋白系统的活细胞内Jun mRNA定位初探

Jun mRNA imaging in living cells based on MS2 coat protein system

目的检测细胞中Jun mRNA在活细胞内的定位。方法设计引物,构建融合MS2茎环结构串联重复的Jun表达载体(pcDNA3.1-Jun-MS2-48X)。转染细胞后,绿色荧光蛋白(GFP)-MS2融合蛋白可与MS2茎环串联重复结构结合,通过荧光显微镜观察GFP的细胞定位,能够间接确定Jun mRNA在细胞内的定位。结果成功构建了pcDNA3.1-Jun-MS2-48X表达载体,经荧光显微镜观察,能够看到明显的绿色荧光,并且在细胞内聚集。结论实现了在细胞内实时观察Jun mRNA,为在单细胞水平上Jun mRNA的定位提供了新的技术方法。

Objective To detect localization of Jun mRNA in living eukaryotic cells. Methods The system utilized the high-affinity interaction between RNA bacteriophage coat protein MS2 and its cognate binding stem loop. By constructing pcDNA3.1-Jun-MS2-48X vector which contained the Jun cDNA and 48 repeats of MS2 binding sites, the Jun mRNA could be fluorescently labeled and visualized by the interaction of GFP-MS2 fusion protein with the stem loop structure of the Jun- MS2-48X transcripts. Results pcDNA3.1-Jun-MS2-g8X Vector was constructed successfully. The localization of GFP, hence the in vivo imaging of Jun mRNA,were determined by the fluorescence microscopy. Conclusion The Jon mRNA is successfully imaged in living HeLa cells, which indicates that the GFP-MS2ztarget mRNA- MS2 binding stem loop system is a robust and effective way to image mRNA in living cells.