Using nucleofection of siRNA constructs for knockdown of primary cilia in P19.CL6 cancer stem cell differentiation into cardiomyocytes.
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.
Clement
Christian A
CA
Department of Biology, Section of Cell and Developmental Biology, University of Copenhagen, DK-2100 Copenhagen OE, Denmark.
Larsen
Lars A
LA
Christensen
Søren T
ST
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D045744:Cell Line, Tumor; D002923:Cilia / Q000502:physiology*; D055785:Gene Knockdown Techniques / Q000295:instrumentation / Q000379:methods; D053823:Hedgehog Proteins / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology / Q000502:physiology*; D014411:Neoplastic Stem Cells / Q000502:physiology / Q000648:ultrastructure; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection / Q000295:instrumentation / Q000379:methods
D015415:Biomarkers; D053823:Hedgehog Proteins; D034741:RNA, Small Interfering