Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
Stem Cells Dev. 2012 Nov 1;21(16):2981-6. doi: 10.1089/scd.2012.0065. Epub 2012 Jun 11.
Recent discovery of a method for derivation and culture of germline-competent rat pluripotent stem cells (PSCs) enables generation of transgenic rats or knock-out rats via genetic modification of such PSCs. This opens the way to use rats, as is routine in mice, for analyses of gene functions or physiological features. In mouse or human, one widely used technique to express a gene of interest stably and ubiquitously is to insert that gene into the Rosa26 locus via gene targeting of PSCs. Rosa26 knock-in mice conditionally expressing a reporter or a toxin gene have contributed to tracing or ablation of specific cell lineages. We successfully identified a rat orthologue of the mouse Rosa26 locus. Insertion of tdTomato, a variant of red fluorescent protein, into the Rosa26 locus of PSCs of various rat strains allows ubiquitous expression of tdTomato. Through germline transmission of one Rosa26-tdTomato knock-in embryonic stem cell line, we also obtained tdTomato knock-in rats. These expressed tdTomato ubiquitously throughout their bodies, which indicates that the rat Rosa26 locus conserves functions of its orthologues in mouse and human. The new tools described here (targeting vectors, knock-in PSCs, and rats) should be useful for a variety of research using rats.
最近发现了一种衍生和培养具有生殖系能力的大鼠多能干细胞（PSCs）的方法，这使得通过对这些 PSCs 进行基因修饰来生成转基因大鼠或敲除大鼠成为可能。这为使用大鼠开辟了道路，就像在小鼠中那样，用于分析基因功能或生理特征。在小鼠或人类中，一种广泛使用的技术是通过 PSCs 的基因靶向将感兴趣的基因插入 Rosa26 基因座，以稳定和普遍表达该基因。条件表达报告基因或毒素基因的 Rosa26 敲入小鼠有助于追踪或消融特定的细胞谱系。我们成功鉴定了小鼠 Rosa26 基因座的大鼠同源物。将红色荧光蛋白变体 tdTomato 插入各种大鼠品系的 PSCs 的 Rosa26 基因座中，可以实现 tdTomato 的普遍表达。通过一条 Rosa26-tdTomato 敲入胚胎干细胞系的种系传递，我们还获得了 tdTomato 敲入大鼠。这些大鼠在其体内普遍表达 tdTomato，这表明大鼠的 Rosa26 基因座保留了其在小鼠和人类中的同源物的功能。本文描述的新工具（靶向载体、敲入 PSCs 和大鼠）应该对使用大鼠进行的各种研究有用。
