Lentiviral shRNA Screen to Identify Epithelial Integrity Regulating Genes in MCF10A 3D Culture
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Author:
Date:
2016-12-05
[Abstract] MCF10A 3D culture system provides a reductionist model of glandular mammary epithelium which is widely used to study development of glandular architecture, the role of cell polarity and epithelial integrity in control of epithelial cell functions, and mechanisms of breast cancer. Here we describe how to use shRNA screening approach to identify critical cell pathways that couple epithelial structure to individual cell based responses such as cell cycle exit and apoptosis. These studies will help to interrogate genetic changes critical for early breast tumorigenesis. The protocol describes a ...
[摘要] MCF10A 3D文化系统提供了腺体乳腺上皮的还原剂模型,其广泛用于研究腺体结构的发育,细胞极性和上皮完整性在上皮细胞功能的控制中的作用以及乳腺癌的机制。在这里我们描述如何使用shRNA筛选方法来识别关键细胞通路,夫妇上皮结构到个别细胞的反应,如细胞周期退出和凋亡。这些研究将有助于询问对早期乳腺肿瘤发生至关重要的遗传变化。该协议描述了设计用于靶向上皮完整性的慢病毒shRNA构建体的文库和用于悬浮MCF10A培养物的慢病毒转导的高效方法。此外,提供的协议设置MCF10A 3D文化在Matrigel的形态和细胞反应研究通过结构化照明和共聚焦显微镜分析免疫染色的三维结构。 关键字: 3D文化,MCF10A,shRNA,上皮完整性,免疫荧光染色,3D成像,形态测量分析 [背景] 上皮细胞形成高度组织的组织结构,其提供物理支持和用于协调细胞信号传导的结构化支架。跨上皮结构的这种协调的信号传导对于上皮生物学是基本的;使得上皮细胞在调节器官大小,形状,功能和基于个体细胞的应答中的动态联合作用(Roignot等人,2013; Shamir和Ewald,2014)。上皮信号传导的联合指挥还提供了一种强有力的肿瘤抑制机制,通过将外部和内部有丝分裂信号门控到静止的上皮组织(Partanen等人,2013; Rejon等人 ...
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Activation of ER-regulated Fusion Proteins In Vivo
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Author:
Date:
2011-12-20
[Abstract] Regulatable protein fusions can be made by attaching the hormone binding domain of the estrogen receptor to the N or C-terminus of a protein of interest. Activation of such ER-fusion proteins in vivo can be achieved by daily administration of a synthetic ligand, 4-hydroxytamoxifen (4OHT), via intraperitoneal (i.p.) injection.
[摘要] 可调节的蛋白融合可以通过贴附雌激素受体的激素结合区域到所感兴趣的蛋白的N或C末端来制备。在生物体内的这种ER-融合蛋白的活化可以每天通过腹腔内注射一种合成的配体物质 4-羟基它莫西芬来完成。
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