(*contributed equally to this work) 发布: 2018年03月20日第8卷第6期 DOI: 10.21769/BioProtoc.2773 浏览次数: 9838
评审: Ralph Thomas BoettcherChris TibbittMurugappan Sathappa
Abstract
The primary cilium is a non-motile sensory organelle whose assembly and disassembly are closely associated with cell cycle progression. The primary cilium is elongated from the basal body in quiescent cells and is resorbed as the cells re-enter the cell cycle. Dysregulation of ciliary dynamics has been linked with ciliopathies and other human diseases. The in vitro serum-stimulated ciliary assembly/disassembly assay has gained popularity in addressing the functions of the protein-of-interest in ciliary dynamics. Here, we describe a well-tested protocol for transfecting human retinal pigment epithelial cells (RPE-1) and performing ciliary assembly/disassembly assays on the transfected cells.
Keywords: Primary cilium (初级纤毛)Background
Primary cilia are hair-like sensory organelles that appear at the G0/G1 phase, and are disassembled prior to the S phase of the cell cycle (Tucker et al., 1979). Previous studies have confirmed that certain non-transformed cell types (i.e., RPE-1 cells, 3T3 fibroblasts, and mouse embryonic fibroblasts [MEFs]) can be starved to induce quiescence and ciliary formation. Subsequent re-addition of serum triggers biphasic ciliary resorption, which peaks at 2 h and 24 h following stimulation (Tucker et al., 1979; Li et al., 2011). This phenomenon lays the foundation for the serum-stimulated ciliary assembly/disassembly assay commonly used in the literature to identify proteins involved in ciliary assembly and disassembly (Pugacheva et al., 2007; Saito et al., 2017). MEFs derived from transgenic mice (in which the gene-of-interest is deleted) are often used to investigate the dynamic role of a given protein in the ciliary assembly/disassembly assays. When the specific MEF types are not accessible, one may modify the expression level of the targeted protein in naive RPE-1 cells (or 3T3 or MEFs) using transfection of cDNA or short hairpin RNA. We describe the protocol of these procedures that are routinely carried out in the lab. To unambiguously identify the cell autonomous effect on ciliary assembly or disassembly in the transfected cells, we typically ‘tag’ the transfected cells with green fluorescence via the expressed GFP or GFP-fusion protein.
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版权信息
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Saito, M., Sakaji, K., Otsu, W. and Sung, C. (2018). Ciliary Assembly/Disassembly Assay in Non-transformed Cell Lines. Bio-protocol 8(6): e2773. DOI: 10.21769/BioProtoc.2773.
分类
发育生物学 > 细胞生长和命运决定 > 增殖
发育生物学 > 细胞信号传导 > 配体
细胞生物学 > 基于细胞的分析方法 > 细胞器能动性
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