Haim-Vilmovsky Liora, Gerst Jeffrey E
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
Methods Mol Biol. 2011;714:237-47. doi: 10.1007/978-1-61779-005-8_15.
Localized mRNA translation is involved in cell-fate determination, polarization, and morphogenesis in eukaryotes. While various tools are available to examine mRNA localization, no easy and quick method has allowed for the visualization of endogenously expressed mRNAs in vivo. We describe a simple method (m-TAG) for PCR-based chromosomal gene tagging that uses homologous recombination to insert binding sites for the RNA-binding MS2 coat protein (MS2-CP) between the coding region and 3'-untranslated region of any yeast gene. Upon co-expression of MS2-CP fused with GFP, specific endogenously expressed mRNAs can be visualized in vivo for the first time. This method allows for the easy examination of mRNA localization using fluorescence microscopy and leaves the yeast cells amenable for further genetic analysis.
局部mRNA翻译参与真核生物的细胞命运决定、极化和形态发生。虽然有各种工具可用于检测mRNA定位，但尚无简单快速的方法能够在体内可视化内源性表达的mRNA。我们描述了一种基于PCR的染色体基因标记的简单方法（m-TAG），该方法利用同源重组在任何酵母基因的编码区和3'非翻译区之间插入RNA结合MS2外壳蛋白（MS2-CP）的结合位点。当与GFP融合的MS2-CP共表达时，首次能够在体内可视化特定的内源性表达的mRNA。该方法允许使用荧光显微镜轻松检测mRNA定位，并使酵母细胞便于进行进一步的遗传分析。
