In vivo Imaging and differential localization of lipid-modified GFP-variant fusions in embryonic stem cells and mice
In vivo Imaging and differential localization of lipid-modified GFP-variant fusions in embryonic stem cells and mice
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DOI：
10.1002/dvg.20203
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发表时间：
2006-04-01
期刊：
影响因子：
1.5
通讯作者：
Hadjantonakis, Anna-Katerina
中科院分区：
文献类型：
--
作者：
Rhee, Jerry M.;Pirity, Melinda K.;Hadjantonakis, Anna-Katerina
活细胞行为的可视化与小鼠遗传学的力量相结合，代表了理解哺乳动物胚胎发育，稳态和疾病进展机制的重要一步。遗传编码的荧光蛋白报告基因的可用性，结合改进的光学成像方式，导致我们的能力，在体内检查细胞的进步。我们开发了一系列脂质修饰的荧光蛋白融合体，其靶向并标记分泌途径和质膜，并且适合用于小鼠。在这里，我们报告了两个品系的小鼠，每个表达光谱不同的脂质修饰的GFP变体荧光蛋白融合的一代。CAG：：GFP-GPI菌株表现出糖基-磷脂酰肌醇-标记的绿色荧光蛋白（GFP）融合体的广泛表达，而CAG：：myr-Venus菌株表现出肉豆蔻酰-Venus黄色荧光蛋白融合体的广泛表达。活的转基因胚胎干细胞（ES），活的或固定的胚胎和出生后组织的成像表明，糖基磷脂酰肌醇和肉豆蔻酰标记的GFP-变体融合蛋白的目标，并作为质膜的标记。此外，我们的数据表明，这两个脂质修饰的蛋白融合动态靶向重叠以及不同的细胞内富含脂质的隔室。这些转基因菌株不仅代表了细胞形态和质膜动力学的高对比度报告，而且还可以用作脂质定位的体内传感器。此外，将这些报告与小鼠突变体的研究相结合，将是理解正常和突变背景下形态发生的细胞间和细胞内行为的一个进步。
The visualization of live cell behaviors operating in situ combined with the power of mouse genetics represents a major step toward understanding the mechanisms regulating embryonic development, homeostasis, and disease progression in mammals. The availability of genetically encoded fluorescent protein reporters, combined with improved optical imaging modalities, have led to advances in our ability to examine cells in vivo. We developed a series of lipid-modified fluorescent protein fusions that are targeted to and label the secretory pathway and the plasma membrane, and that are amenable for use in mice. Here we report the generation of two strains of mice, each expressing a spectrally distinct lipid-modified GFP-variant fluorescent protein fusion. The CAG::GFP-GPI strain exhibited widespread expression of a glycosyl-phosphatidylinositol-tagged green fluorescent protein (GFP) fusion, while the CAG::myr-Venus strain exhibited widespread expression of a myristoyl-Venus yellow fluorescent protein fusion. Imaging of live transgenic embryonic stem (ES) cells, either live or fixed embryos and postnatal tissues demonstrated that glycosyllphosphatidyl inositol- and myristoyll-tagged GFP-variant fusion proteins are targeted to and serve as markers of the plasma membrane. Moreover, our data suggest that these two lipid-modified protein fusions are dynamically targeted both to overlapping as well as distinct lipid-enriched compartments within cells. These transgenic strains not only represent high-contrast reporters of cell morphology and plasma membrane dynamics, but also may be used as in vivo sensors of lipid localization. Furthermore, combining these reporters with the study of mouse mutants will be a step forward in understanding the inter- and intracellular behaviors underlying morphogenesis in both normal and mutant contexts.