Department of Genetics, North Carolina State University, Raleigh, NC 27695, USA.
Plant J. 2011 May;66(4):712-23. doi: 10.1111/j.1365-313X.2011.04524.x. Epub 2011 Mar 9.
One of the most information-rich aspects of gene functional studies is characterization of gene expression profiles at cellular resolution, and subcellular localization of the corresponding proteins. These studies require visualization of the endogenous gene products using specific antibodies, or, more commonly, generation of whole-gene translational fusions with a reporter gene such as a fluorescent protein. To facilitate the generation of such translational fusions and to ensure that all cis-regulatory sequences are included, we have used a bacterial homologous recombination system (recombineering) to insert fluorescent protein tags into genes of interest harbored by transformation-competent bacterial artificial chromosomes (TACs). This approach has several advantages compared to other classical strategies. First, the researcher does not have to guess what the regulatory sequences of a gene are, as tens of thousands of base pairs flanking the gene of interest can be included in the construct. Second, because the genes of interest are not amplified by PCR, there are practically no limits to the size of a gene that can be tagged. Third, there are no restrictions on the location in which the fluorescent protein can be inserted, as the position is determined by sequence homology with the recombination primers. Finally, all of the required strains and TAC clones are publically available, and the experimental procedures described here are simple and robust. Thus, we suggest that recombineering-based gene tagging should be the gold standard for gene expression studies in Arabidopsis.
基因功能研究最具信息量的方面之一是在细胞分辨率下对基因表达谱进行特征描述，以及相应蛋白质的亚细胞定位。这些研究需要使用特定的抗体对内源性基因产物进行可视化，或者更常见的是，通过与报告基因（如荧光蛋白）的全基因翻译融合来生成。为了促进这种翻译融合的产生，并确保包含所有顺式调控序列，我们使用细菌同源重组系统（重组酶）将荧光蛋白标签插入转化能力强的细菌人工染色体（TAC）中感兴趣的基因中。与其他经典策略相比，这种方法有几个优点。首先，研究人员不必猜测基因的调控序列是什么，因为可以将感兴趣基因侧翼的数万碱基对包含在构建体中。其次，由于感兴趣的基因不是通过 PCR 扩增的，因此可以标记的基因大小实际上没有限制。第三，荧光蛋白插入的位置不受限制，因为其位置由与重组引物的序列同源性决定。最后，所有必需的菌株和 TAC 克隆都是公开可用的，并且这里描述的实验程序简单且稳健。因此，我们建议基于重组酶的基因标记应该成为拟南芥基因表达研究的黄金标准。
