A functional genomic analysis of cell morphology using RNA interference.
A functional genomic analysis of cell morphology using RNA interference.
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DOI：
10.1186/1475-4924-2-27
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发表时间：
2003
影响因子：
--
通讯作者：
Perrimon N
中科院分区：
文献类型：
--
作者：
Kiger AA;Baum B;Jones S;Jones MR;Coulson A;Echeverri C;Perrimon N
后生动物细胞形状的多样性受到动态细胞骨架网络的影响。随着rna干扰（RNAi）技术的出现，现在可以系统地筛选控制特定细胞生物学过程的基因，包括那些产生不同形态所需的基因。我们改编了果蝇细胞培养中现有的RNAi技术，用于高通量筛选，以便对细胞形态发生进行全面的遗传解剖。为了确定两种形态不同的细胞系的特征形状的基因，我们在每个细胞系中使用一组针对994种预测细胞形状调节因子的双链rna （dsRNAs）进行了RNAi筛选。使用自动荧光显微镜观察肌动蛋白细丝、微管和DNA，我们检测到160个基因的形态表型，其中三分之一以前没有在体内表征过。具有相似表型的基因对应于控制细胞骨架组织和细胞形状的途径的已知组分，这使我们提出了以前未表征的基因的类似功能。此外，我们能够使用co-RNAi筛选发现在特定途径中起作用的基因，以鉴定Pten dsRNA诱导的细胞形状变化的dsRNA抑制因子。使用RNAi，我们鉴定了影响两种不同细胞类型的细胞骨架组织和形态的基因。一些基因在两种细胞类型中表现出相似的RNAi表型，而其他基因似乎具有细胞类型特异性功能，部分反映了用于产生圆形或扁平细胞形态的不同机制。
The diversity of metazoan cell shapes is influenced by the dynamic cytoskeletal network. With the advent of RNA-interference (RNAi) technology, it is now possible to screen systematically for genes controlling specific cell-biological processes, including those required to generate distinct morphologies. We adapted existing RNAi technology in Drosophila cell culture for use in high-throughput screens to enable a comprehensive genetic dissection of cell morphogenesis. To identify genes responsible for the characteristic shape of two morphologically distinct cell lines, we performed RNAi screens in each line with a set of double-stranded RNAs (dsRNAs) targeting 994 predicted cell shape regulators. Using automated fluorescence microscopy to visualize actin filaments, microtubules and DNA, we detected morphological phenotypes for 160 genes, one-third of which have not been previously characterized in vivo. Genes with similar phenotypes corresponded to known components of pathways controlling cytoskeletal organization and cell shape, leading us to propose similar functions for previously uncharacterized genes. Furthermore, we were able to uncover genes acting within a specific pathway using a co-RNAi screen to identify dsRNA suppressors of a cell shape change induced by Pten dsRNA. Using RNAi, we identified genes that influence cytoskeletal organization and morphology in two distinct cell types. Some genes exhibited similar RNAi phenotypes in both cell types, while others appeared to have cell-type-specific functions, in part reflecting the different mechanisms used to generate a round or a flat cell morphology.