Rino José, de Jesus Ana C, Carmo-Fonseca Maria
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028, Lisbon, Portugal.
Methods Mol Biol. 2017;1563:229-242. doi: 10.1007/978-1-4939-6810-7_15.
Single mRNA molecules can be imaged in living cells by a method that consists in genetically inserting binding sites for a bacteriophage protein in the gene of interest. The resulting reporter transgene is then integrated in the genome of cells that express the phage protein fused to a fluorescent tag. Upon transcription, binding of the fluorescent protein to its target sequence makes the RNA visible. With this approach it is possible to track, in real time, the life cycle of a precursor mRNA at the site of transcription in the nucleus and transport of mature mRNA to the cytoplasm. In order to measure the fluorescence associated with individual RNA molecules over time, we developed a semi-automated quantitative image analysis tool termed STaQTool. We describe in detail the implementation and application of the STaQTool software package, which is a generic tool able to process large 4D datasets allowing quantitative studies of different steps in gene expression.
通过一种方法可以在活细胞中对单个信使核糖核酸（mRNA）分子进行成像，该方法包括在感兴趣的基因中通过基因手段插入噬菌体蛋白的结合位点。然后将产生的报告转基因整合到表达与荧光标签融合的噬菌体蛋白的细胞基因组中。转录时，荧光蛋白与其靶序列的结合使RNA可见。通过这种方法，可以实时追踪前体mRNA在细胞核转录位点的生命周期以及成熟mRNA向细胞质的转运。为了随时间测量与单个RNA分子相关的荧光，我们开发了一种称为STaQTool的半自动定量图像分析工具。我们详细描述了STaQTool软件包的实现和应用，它是一个通用工具，能够处理大型4D数据集，从而对基因表达的不同步骤进行定量研究。
