Visualization of cell cycle in mouse embryos with Fucci2 reporter directed by Rosa26 promoter
Visualization of cell cycle in mouse embryos with Fucci2 reporter directed by Rosa26 promoter
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DOI：
10.1242/dev.084111
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发表时间：
2013-01-01
期刊：
影响因子：
4.6
通讯作者：
Fujimori, Toshihiko
中科院分区：
文献类型：
--
作者：
Abe, Takaya;Sakaue-Sawano, Asako;Fujimori, Toshihiko
Fucci技术可以通过双色成像区分G（1）和S/G（2）/M期的活细胞。该技术依赖于泛素化介导的蛋白水解，表达Fucci的转基因小鼠提供了一个强大的模型系统，用于研究细胞周期和发育的协调。最初使用CAG启动子产生小鼠;分别构建表达分别发射橙子（mKO 2）和绿色（mAG）荧光的G（1）和S/G（2）/M期探针的品系。然而，由于CAG启动子的细胞类型偏好强度以及随机转基因的位置效应，我们注意到Fucci表达水平的一些变异性。为了更可靠地控制细胞周期探针的表达，我们使用不同的遗传方法来创建具有Fucci 2和Rosa 26转录机制的两种类型的报告小鼠系。Fucci 2是最近开发的Fucci衍生物，其发射红色（mCherry）和绿色（mVenus）荧光，并且提供比Fucci更好的颜色对比度。一个新的转基因株系R26 p-Fucci 2利用Rosa 26启动子并在单个转基因中携带G（1）和S/G（2）/M期探针以保持它们的共遗传。在另一种R26 R-Fucci 2方法中，两种探针有条件地掺入Rosa 26基因座中。因此，Cre介导的loxP重组技术允许研究人员设计细胞类型特异性Fucci 2表达。通过使用R26 p-Fucci 2和R26-Fucci 2进行延时成像实验，其中R26 R-Fucci 2经历了种系loxP重组，我们证明了这些小鼠报告基因在体内研究细胞周期行为方面的巨大前景。
Fucci technology makes possible the distinction between live cells in the G(1) and S/G(2)/M phases by dual-color imaging. This technology relies upon ubiquitylation-mediated proteolysis, and transgenic mice expressing Fucci provide a powerful model system with which to study the coordination of the cell cycle and development. The mice were initially generated using the CAG promoter; lines expressing the G(1) and S/G(2)/M phase probes that emitted orange (mKO2) and green (mAG) fluorescence, respectively, were separately constructed. Owing to cell type-biased strength of the CAG promoter as well as the positional effects of random transgenesis, however, we noticed some variability in Fucci expression levels. To control more reliably the expression of cell cycle probes, we used different genetic approaches to create two types of reporter mouse lines with Fucci2 and Rosa26 transcriptional machinery. Fucci2 is a recently developed Fucci derivative, which emits red (mCherry) and green (mVenus) fluorescence and provides better color contrast than Fucci. A new transgenic line, R26p-Fucci2, utilizes the Rosa26 promoter and harbors the G(1) and S/G(2)/M phase probes in a single transgene to preserve their co-inheritance. In the other R26R-Fucci2 approach, the two probes are incorporated into Rosa26 locus conditionally. The Cre-mediated loxP recombination technique thus allows researchers to design cell-type-specific Fucci2 expression. By performing time-lapse imaging experiments using R26p-Fucci2 and R26-Fucci2 in which R26R-Fucci2 had undergone germline loxP recombination, we demonstrated the great promise of these mouse reporters for studying cell cycle behavior in vivo.