Kakitani Makoto, Oshima Takeshi, Horikoshi Kaori, Yoshitome Tetsuo, Ueda Akiko, Kajikawa Miwa, Iba Yumi, Ozone Yoshinao, Ijima Yuki, Yoshino Tohko, Itoh Mikiko, Seki Sachiko, Aoki Ayako, Ishihara Toshie, Shionoya Michiyo, Makino Utako, Kitada Rina, Ohguma Atsuko, Ohta Takami, Yoshida Yoshimasa, Kudoh Hiroe, Hanaoka Kazunori, Sibuya Kazunori, Ishida Isao, Kakeda Minoru, Yagi Mikio, Yoneya Takashi, Tomizuka Kazuma
Pharmaceutical Research Laboratory, Pharmaceutical Division, Kirin Brewery Co. Ltd. 3 Miyahara-cho, Takasaki-shi, Gunma 370-1295, Japan.
Nucleic Acids Res. 2005 May 24;33(9):e85. doi: 10.1093/nar/gni083.
A major challenge of the post-genomic era is the functional characterization of anonymous open reading frames (ORFs) identified by the Human Genome Project. In this context, there is a strong requirement for the development of technologies that enhance our ability to analyze gene functions at the level of the whole organism. Here, we describe a rapid and efficient procedure to generate transgenic chimaeric mice that continuously secrete a foreign protein into the systemic circulation. The transgene units were inserted into the genomic site adjacent to the endogenous immunoglobulin (Ig) kappa locus by homologous recombination, using a modified mouse embryonic stem (ES) cell line that exhibits a high frequency of homologous recombination at the Igkappa region. The resultant ES clones were injected into embryos derived from a B-cell-deficient host strain, thus producing chimaerism-independent, B-cell-specific transgene expression. This feature of the system eliminates the time-consuming breeding typically implemented in standard transgenic strategies and allows for evaluating the effect of ectopic transgene expression directly in the resulting chimaeric mice. To demonstrate the utility of this system we showed high-level protein expression in the sera and severe phenotypes in human EPO (hEPO) and murine thrombopoietin (mTPO) transgenic chimaeras.
后基因组时代的一个主要挑战是对人类基因组计划所识别的无名开放阅读框（ORF）进行功能表征。在此背景下，迫切需要开发能够增强我们在全生物体水平分析基因功能能力的技术。在此，我们描述了一种快速有效的方法来生成能持续向体循环分泌外源蛋白的转基因嵌合小鼠。通过同源重组，将转基因单元插入到与内源性免疫球蛋白（Ig）κ基因座相邻的基因组位点，使用的是一种经修饰的小鼠胚胎干细胞（ES）系，该细胞系在Igκ区域表现出高频同源重组。将所得的ES克隆注射到源自B细胞缺陷宿主品系的胚胎中，从而产生不依赖嵌合现象的B细胞特异性转基因表达。该系统的这一特性消除了标准转基因策略中通常实施的耗时育种过程，并允许直接在所得的嵌合小鼠中评估异位转基因表达的效果。为了证明该系统的实用性，我们在人促红细胞生成素（hEPO）和小鼠血小板生成素（mTPO）转基因嵌合体的血清中展示了高水平的蛋白表达以及严重的表型。
