在 mESC 中进行高通量筛选,以确定信号传导、内吞和多能性之间的交叉联系。

IF 3.3 3区 生物学 Q3 CELL BIOLOGY
Ridim D. Mote, Mahak Tiwari, Narayana Yadavalli, Raghav Rajan, Deepa Subramanyam
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引用次数: 0

摘要

胚胎干细胞的命运受多种细胞过程的调控。最近,内吞过程被认为在维持小鼠胚胎干细胞的自我更新和多能性方面发挥作用。先前的一项基于 siRNA 的筛选研究考察了内吞机制核心部件在维持胚胎干细胞多能性中的功能,发现了凝集素介导的内吞作用的关键作用。其他一些参与关键信号通路的蛋白质也被证明既能调节内吞,也能受内吞调节。我们从《京都基因与基因组百科全书》(KEGG)和《基因本体论》(GO)中整理出了与 "内吞 "相关的1141个基因列表,其中不包括之前已经研究过的基因,并利用Oct4启动子驱动的绿色荧光蛋白水平研究了敲除这些基因对小鼠胚胎干细胞(mESCs)多能性的影响。我们利用高通量筛选,然后使用基于MATrix LABoratory(MATLAB)的自动分析管道,评估GFP荧光的变化,作为ESC多能性的读数。通过这一筛选,我们发现了许多基因,其中许多迄今与干细胞多能性无关,这些基因敲除后会导致GFP荧光显著增加或减少。我们还对其中一些基因进行了验证。我们介绍了一种工作流程,旨在鉴定参与信号通路的基因,这些通路可受内吞调节,并影响干细胞的多能性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
A high-throughput screen in mESCs to identify the cross-talk between signaling, endocytosis, and pluripotency

Embryonic stem cell fate is regulated by various cellular processes. Recently, the process of endocytosis has been implicated in playing a role in the maintenance of self-renewal and pluripotency of mouse embryonic stem cells. A previous siRNA-based screen interrogated the function of core components of the endocytic machinery in maintaining the pluripotency of embryonic stem cells, revealing a crucial role for clathrin mediated endocytosis. A number of other proteins involved in key signaling pathways have also been shown to both regulate and be regulated by endocytosis. We collated a list of 1141 genes in connection to the term “endocytosis” from Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO), excluding those previously interrogated, and examined the effect of their knockdown on the pluripotency of mouse embryonic stem cells (mESCs) using levels of green fluorescent protein driven by the Oct4 promoter. We used high-throughput screening followed by an automated MATrix LABoratory (MATLAB)-based analysis pipeline and assessed changes in GFP fluorescence as a readout for ESC pluripotency. Through this screen we identified a number of genes, many hitherto not associated with stem cell pluripotency, which upon knockdown either resulted in a significant increase or decrease of GFP fluorescence. We further present validation for some of these hits. We present a workflow aimed to identify genes involved in signaling pathways which can be regulated by endocytosis, and that affect the pluripotency of ESCs.

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来源期刊
Cell Biology International
Cell Biology International 生物-细胞生物学
CiteScore
7.60
自引率
0.00%
发文量
208
审稿时长
1 months
期刊介绍: Each month, the journal publishes easy-to-assimilate, up-to-the minute reports of experimental findings by researchers using a wide range of the latest techniques. Promoting the aims of cell biologists worldwide, papers reporting on structure and function - especially where they relate to the physiology of the whole cell - are strongly encouraged. Molecular biology is welcome, as long as articles report findings that are seen in the wider context of cell biology. In covering all areas of the cell, the journal is both appealing and accessible to a broad audience. Authors whose papers do not appeal to cell biologists in general because their topic is too specialized (e.g. infectious microbes, protozoology) are recommended to send them to more relevant journals. Papers reporting whole animal studies or work more suited to a medical journal, e.g. histopathological studies or clinical immunology, are unlikely to be accepted, unless they are fully focused on some important cellular aspect. These last remarks extend particularly to papers on cancer. Unless firmly based on some deeper cellular or molecular biological principle, papers that are highly specialized in this field, with limited appeal to cell biologists at large, should be directed towards journals devoted to cancer, there being very many from which to choose.
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