RNA interference techniques to study epithelial cell adhesion and polarity.
Polarized epithelial cells are characterized by distinct plasma membrane domains and asymmetrical distribution of cell surface proteins and lipids. In vertebrates, tight junctions act as a fence between the apical and basolateral domains. Although many of the key components of the polarity machinery have been identified, their functions in cell polarization and junction formation remain to be determined. With the rapid improvement of the RNA interference (RNAi) technique, it is now possible to silence the expression of these polarity proteins in mammalian cells and to systematically analyze their distinct roles in orchestrating the polarization program. Here we describe approaches to achieve specific gene suppression in MDCK cells, a well-established cell culture model of canine kidney cells. We discuss the potential challenges and problems associated with the RNAi technique and describe basic protocols for suppressing gene expression using a vector-based short hairpin RNA (shRNA) expression system coupled with nucleofection.
Center for Cell Signalling, University of Virginia School of Medicine, Charlottesville, USA.
Xinyu
X
Chen
Ian G
IG
Macara
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000172:deficiency; D018797:Cell Cycle Proteins; D016764:Cell Polarity / Q000502:physiology*; D004285:Dogs; D004847:Epithelial Cells / Q000502:physiology*; D005822:Genetic Vectors; D051379:Mice; D034622:RNA Interference; D019108:Tight Junctions / Q000502:physiology; D014162:Transfection / Q000379:methods
D048868:Adaptor Proteins, Signal Transducing; D015815:Cell Adhesion Molecules; D018797:Cell Cycle Proteins; C115084:Pard3 protein, mouse