Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Pawinskiego 5a, 02-106, Warsaw, Poland and Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland.
Nucleic Acids Res. 2013 Sep;41(17):e167. doi: 10.1093/nar/gkt650. Epub 2013 Jul 27.
DT40 cells derived from chicken B lymphocytes exhibit exceptionally high homologous recombination rates. Therefore, they can be used as a convenient tool and model for gene targeting experiments. However, lack of efficient cloning strategies, protein purification protocols and a well annotated protein database limits the utility of these cells for proteomic studies. Here we describe a fast and inexpensive experimental pipeline for protein localization, quantification and mass spectrometry-based interaction studies using DT40 cells. Our newly designed set of pQuant vectors and a sequence- and ligation-independent cloning (SLIC) strategy allow for simple and efficient generation of gene targeting constructs, facilitating homologous-recombination-based protein tagging on a multi-gene scale. We also report proof of principle results using the key proteins involved in RNA decay, namely EXOSC8, EXOSC9, CNOT7 and UPF1.
从鸡 B 淋巴细胞衍生而来的 DT40 细胞表现出异常高的同源重组率。因此，它们可以作为基因靶向实验的便捷工具和模型。然而，缺乏有效的克隆策略、蛋白质纯化方案和注释良好的蛋白质数据库限制了这些细胞在蛋白质组学研究中的应用。在这里，我们描述了一种使用 DT40 细胞进行蛋白质定位、定量和基于质谱的相互作用研究的快速、廉价的实验方案。我们新设计的一组 pQuant 载体和一种序列和连接独立克隆（SLIC）策略允许简单、高效地生成基因靶向构建体，从而在多基因水平上促进基于同源重组的蛋白质标记。我们还报告了使用涉及 RNA 降解的关键蛋白质（即 EXOSC8、EXOSC9、CNOT7 和 UPF1）的原理验证结果。
