Kim Jung-Sik, Bonifant Challice, Bunz Fred, Lane William S, Waldman Todd
Lombardi Comprehensive Cancer Center, Georgetown University School of Medicine, Washington, DC 20057, USA.
Nucleic Acids Res. 2008 Nov;36(19):e127. doi: 10.1093/nar/gkn566. Epub 2008 Sep 10.
Epitope tagging is a powerful and commonly used approach for studying the physical properties of proteins and their functions and localization in eukaryotic cells. In the case of Saccharomyces cerevisiae, it has been possible to exploit the high efficiency of homologous recombination to tag proteins by modifying their endogenous genes, making it possible to tag virtually every endogenous gene and perform genome-wide proteomics experiments. However, due to the relative inefficiency of homologous recombination in cultured human cells, epitope-tagging approaches have been limited to ectopically expressed transgenes, with the attendant limitations of their nonphysiological transcriptional regulation and levels of expression. To overcome this limitation, a modification and extension of adeno-associated virus-mediated human somatic cell gene targeting technology is described that makes it possible to simply and easily create an endogenous epitope tag in the same way that it is possible to knock out a gene. Using this approach, we have created and validated human cell lines with epitope-tagged alleles of two cancer-related genes in a variety of untransformed and transformed human cell lines. This straightforward approach makes it possible to study the physical and biological properties of endogenous proteins in human cells without the need for specialized antibodies for individual proteins of interest.
表位标签法是一种用于研究蛋白质物理性质、功能及其在真核细胞中定位的强大且常用的方法。就酿酒酵母而言，利用同源重组的高效性，通过修饰其内源基因来对蛋白质进行标签标记成为可能，这使得几乎可以对每个内源基因进行标签标记并开展全基因组蛋白质组学实验。然而，由于在培养的人类细胞中同源重组效率相对较低，表位标签法一直局限于异位表达的转基因，伴随着非生理性转录调控和表达水平等相关局限性。为克服这一局限，本文描述了腺相关病毒介导的人类体细胞基因靶向技术的一种改进和扩展，该技术使得能够以与敲除基因相同的方式简单轻松地创建一个内源表位标签。利用这种方法，我们在多种未转化和转化的人类细胞系中创建并验证了带有两个癌症相关基因表位标签化等位基因的人类细胞系。这种直接的方法使得无需针对感兴趣的单个蛋白质制备专门抗体就能研究人类细胞中内源蛋白质的物理和生物学特性。
