Stem Cell and Developmental Biology, Genome Institute of Singapore, 60 Biopolis Street, Singapore, 138672, Singapore.
Transgenic Res. 2012 Apr;21(2):293-301. doi: 10.1007/s11248-011-9531-9. Epub 2011 Jul 6.
Gene expression is usually studied at the transcript level rather than at the protein level due to the lack of a specific and sensitive antibody. A way to overcome this is to fuse to the protein of interest an immunoreactive tag that has well-characterized antibodies. This epitope tagging approach is often used for in vitro experiments but for in vivo studies, the success rate of protein tagging has not been extensively analyzed and our study seeks to cover the void. A small epitope, hemaglutinin derived from the influenza virus was used to tag a transcription factor, Sox5 at the N-terminal via homologous recombination in the mouse. Sox5 is part of the Sry-related high-mobility-group box gene family and plays multiple roles in essential biological processes. Understanding of its molecular function in relation to its biological roles remains incomplete. In our study, we show that the longer isoform of Sox5 can be tagged endogenously with hemaglutinin without affecting its biological function in vivo. The tagged protein is easily and specifically detected with an anti-hemaglutinin antibody using immunohistochemistry with its expression matching the endogenous expression of Sox5. Immunoprecipitation of Sox5 was also carried out successfully using an anti-hemaglutinin antibody. The transgenic line generated from this study is predicted to be useful for future experiments such as co-immunoprecipitation and chromatin immunoprecipitation, allowing the further understanding of Sox5. Lastly, this approach can be easily employed for the investigation of other transcription factors and proteins in vivo to overcome technical limitations such as antibody cross-reactivity and to perform isoform-specific studies.
由于缺乏特异性和敏感性抗体，基因表达通常在转录水平上进行研究，而不是在蛋白质水平上进行研究。一种克服这个问题的方法是将感兴趣的蛋白质与具有良好特征的抗体的免疫反应标签融合。这种表位标记方法常用于体外实验，但对于体内研究，蛋白质标记的成功率尚未广泛分析，我们的研究旨在填补这一空白。我们使用源自流感病毒的小表位血凝素通过同源重组在小鼠中标记转录因子 Sox5 的 N 端。Sox5 是 Sry 相关高迁移率族盒基因家族的一部分，在许多重要的生物过程中发挥多种作用。尽管其分子功能与其生物学作用之间的关系尚未完全了解，但我们的研究表明，Sox5 的较长同工型可以通过内源性血凝素标记而不影响其体内的生物学功能。使用抗血凝素抗体通过免疫组织化学可以轻松且特异性地检测到标记的蛋白质，其表达与 Sox5 的内源性表达相匹配。使用抗血凝素抗体也成功地进行了 Sox5 的免疫沉淀。从这项研究中产生的转基因系预计将对未来的实验（如共免疫沉淀和染色质免疫沉淀）有用，从而进一步了解 Sox5。最后，这种方法可以很容易地用于研究其他转录因子和蛋白质在体内，以克服抗体交叉反应等技术限制，并进行同工型特异性研究。
