A novel transgenic chimaeric mouse system for the rapid functional evaluation of genes encoding secreted proteins
A novel transgenic chimaeric mouse system for the rapid functional evaluation of genes encoding secreted proteins
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DOI：
10.1093/nar/gni083
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发表时间：
2005-01-01
影响因子：
14.9
通讯作者：
Tomizuka, K
中科院分区：
文献类型：
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作者：
Kakitani, M;Oshima, T;Tomizuka, K
后基因组时代的一个主要挑战是人类基因组计划确定的匿名开放阅读框（orf）的功能表征。在这种情况下，有一个强烈的要求，技术的发展，提高我们的能力，分析基因功能在整个生物体的水平。在这里，我们描述了一个快速和有效的程序，以产生转基因嵌合小鼠，不断分泌外源蛋白进入体循环。利用在Ig kappa区表现出高同源重组频率的修饰小鼠胚胎干（ES）细胞系，通过同源重组将转基因单元插入内源性免疫球蛋白（Ig） kappa位点附近的基因组位点。由此产生的ES克隆被注射到来自B细胞缺陷宿主菌株的胚胎中，从而产生不依赖嵌合的、B细胞特异性的转基因表达。该系统的这一特点消除了在标准转基因策略中典型实施的耗时育种，并允许直接评估异位转基因在最终嵌合小鼠中的表达效果。为了证明该系统的实用性，我们在人类EPO （hEPO）和小鼠血小板生成素（mTPO）转基因嵌合体的血清中显示了高水平的蛋白表达和严重的表型。
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 Ig kappa 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 transgenicstrategiesandallows 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.