Clement Christian A, Larsen Lars A, Christensen Søren T
Department of Biology, Section of Cell and Developmental Biology, University of Copenhagen, DK-2100 Copenhagen OE, Denmark.
Methods Cell Biol. 2009;94:181-97. doi: 10.1016/S0091-679X(08)94009-7. Epub 2009 Dec 23.
Primary cilia assemble as solitary organelles in most mammalian cells during growth arrest and are thought to coordinate a series of signal transduction pathways required for cell cycle control, cell migration, and cell differentiation during development and in tissue homeostasis. Recently, primary cilia were suggested to control pluripotency, proliferation, and/or differentiation of stem cells, which may comprise an important source in regenerative biology. We here provide a method using a P19.CL6 embryonic carcinoma (EC) stem cell line to study the function of the primary cilium in early cardiogenesis. By knocking down the formation of the primary cilium by nucleofection of plasmid DNA with siRNA sequences against genes essential in ciliogenesis (IFT88 and IFT20) we block hedgehog (Hh) signaling in P19.CL6 cells as well as the differentiation of the cells into beating cardiomyocytes (Clement et al., 2009). Immunofluorescence microscopy, western blotting, and quantitative PCR analysis were employed to delineate the molecular and cellular events in cilia-dependent cardiogenesis. We optimized the nucleofection procedure to generate strong reduction in the frequency of ciliated cells in the P19.CL6 culture.
在生长停滞期间，大多数哺乳动物细胞中的初级纤毛作为单个细胞器组装形成，并且被认为在发育过程以及组织内稳态中协调细胞周期控制、细胞迁移和细胞分化所需的一系列信号转导途径。最近，有人提出初级纤毛可控制干细胞的多能性、增殖和/或分化，这可能是再生生物学的一个重要来源。我们在此提供一种使用P19.CL6胚胎癌细胞（EC）系来研究初级纤毛在早期心脏发生中功能的方法。通过用针对纤毛发生中必需基因（IFT88和IFT20）的siRNA序列进行质粒DNA核转染来抑制初级纤毛的形成，我们阻断了P19.CL6细胞中的刺猬（Hh）信号传导以及细胞向跳动心肌细胞的分化（克莱门特等人，2009年）。采用免疫荧光显微镜、蛋白质印迹和定量PCR分析来描绘纤毛依赖心脏发生中的分子和细胞事件。我们优化了核转染程序，以显著降低P19.CL6培养物中有纤毛细胞的频率。
