Single-copy transgenic mice with chosen-site integration
Single-copy transgenic mice with chosen-site integration
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DOI：
10.1073/pnas.93.17.9067
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发表时间：
1996-08-20
影响因子：
11.1
通讯作者：
Smithies, O
中科院分区：
文献类型：
--
作者：
Bronson, SK;Plaehn, EG;Smithies, O
我们描述了一种将转基因引入小鼠生殖系的一般方法，用于比较不同的序列，而不会出现拷贝数和插入位点变化的并发症。该方法使用胚胎干（ES）细胞中的同源重组来产生具有整合到基因组中的选定位置中的转基因的单拷贝的小鼠。为了测试该方法，通过直接选择的同源重组事件，将由鸡β-肌动蛋白启动子或人β-肌动蛋白启动子驱动的单拷贝鼠bcl-2 cDNA直接插入X-连锁次黄嘌呤磷酸核糖基转移酶基因座的5 ′。ES细胞中靶向bcl-2转基因的表达水平在具有相同启动子的独立分离的同源重组体中是相同的，但在不同启动子之间变化。相反，当具有相同（鸡β-肌动蛋白）启动子的bcl-2转基因独立整合在随机插入位点时，它们的表达变化剧烈。两种启动子都指导单拷贝转基因在源自各自靶向ES细胞的小鼠中的广泛表达。在体外和体内，人β-肌动蛋白启动子始终指导比鸡β-肌动蛋白启动子更高水平的转基因表达。
We describe a general way of introducing transgenes into the mouse germ line for comparing different sequences without the complications of variation in copy number and insertion site. The method uses homologous recombination in embryonic stem (ES) cells to generate mice having a single copy of a transgene integrated into a chosen location in the genome. To test the method, a single copy murine bcl-2 cDNA driven by either a chicken beta-actin promoter or a human beta-actin promoter has been inserted immediately 5' to the X-linked hypoxanthine phosphoribosyltransferase locus by a directly selectable homologous recombination event. The level of expression of the targeted bcl-2 transgene in ES cells is identical in independently isolated homologous recombinants having the same promoter yet varies between the different promoters, In contrast, the expression of bcl-2 transgenes having the same (chicken beta-actin) promoter varies drastically when they are independently integrated at random insertion sites. Both promoters direct broad expression of the single-copy transgene in mice derived from the respective targeted ES cells, In vitro and in vivo, the human beta-actin promoter consistently directed a higher level of transgene expression than the chicken beta-actin promoter.