Establishment of Conditional Reporter Mouse Lines at ROSA26 Locus For Live Cell Imaging
Establishment of Conditional Reporter Mouse Lines at ROSA26 Locus For Live Cell Imaging
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DOI：
10.1002/dvg.20753
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发表时间：
2011-07-01
期刊：
影响因子：
1.5
通讯作者：
Fujimori, Toshihiko
中科院分区：
文献类型：
--
作者：
Abe, Takaya;Kiyonari, Hiroshi;Fujimori, Toshihiko
建立了一系列条件报告小鼠系，其中特定细胞器用荧光蛋白标记。研究人员对细胞系中 28 种荧光融合蛋白构建体的亚细胞定位和强度进行了调查，然后选择 16 种构建体来生成小鼠系。将融合 cDNA 插入 ROSA26 基因组基因座中，靠近侧翼为 IoxP 的终止序列，以便当 IoxP 序列与 Cre 重组时，荧光蛋白在无处不在的 ROSA26 转录机制下表达。通过在 E7.5 胚胎中普遍表达融合产物来检查每个报告小鼠系中融合产物的亚细胞定位和强度。发现标记细胞核、线粒体、高尔基体、质膜、微管、肌动蛋白丝和粘着斑的 12 条报告线适合实时成像。细胞核和质膜或高尔基体具有明显的双重染色；获得细胞核和质膜的清晰延时实时图像；用 Not-Cre 在脊索中的 Lyn-Venus 和 H2B-mCherry 系上证实了条件表达。创世纪 49: 579-590, 2011。(C) 2011 Wiley-Liss, Inc.
A series of conditional reporter mouse lines were established in which specific organelles were labeled with fluorescent proteins. Subcellular localization and intensity of 28 fluorescent fusion-protein constructs were surveyed in cell lines, and 16 constructs then were selected to generate mouse lines. The fusion cDNAs were inserted into the ROSA26 genomic locus next to the stop sequences flanked with IoxP so that fluorescent proteins were expressed under the ubiquitous ROSA26 transcriptional machinery when the IoxP sequences were recombined with Cre. The subcellular localization and intensity of the fusion product in each reporter mouse line were examined by ubiquitously expressing them in E7.5 embryos. Twelve reporter lines, that mark nucleus, mitochondria, Golgi apparatus, plasma membrane, microtubule, actin filament, and focal adhesion, were found suitable for live imaging. Distinct double staining was demonstrated for nucleus and plasma membrane or Golgi apparatus; clear time-lapse live images were obtained for nucleus and plasma membranes; conditional expression was confirmed on Lyn-Venus and H2B-mCherry lines in notochord with Not-Cre. genesis 49: 579-590, 2011. (C) 2011 Wiley-Liss, Inc.