title: Using replication defective viruses to analyze membrane trafficking in polarized epithelial cells.
abstract: Epithelial cells in culture, especially once they are polarized, are extremely hard to manipulate by transient transfection methods. The use of replication defective adenoviruses for gene expression or replication defective retroviruses or lentiviruses to express shRNA for gene knockdown provides efficient tools to manipulate gene expression patterns even in hard-to-transfect cell lines. One of the advantages of using defective adenoviruses for gene expression is that once the virus has been generated, it can easily be applied to a wide variety of cells. In addition, replication defective retro- and lentiviruses are used to stably deplete proteins from cell lines, which subsequently may be used for analyzing the polarized surface delivery of receptors that may be expressed using defective adenoviruses. The latter approach is especially useful if the expressed shRNA also encodes GFP for easy assessment of shRNA-expressing cells. Thus the use of defective viruses in epithelial cell research is convenient. This makes a detailed infection protocol a research tool that would be valuable to many laboratories. Here we describe in detail how cells are infected with defective retro- or lentiviruses and subsequently selected for stable gene knockdown. We then describe how these cells may be used for infection with defective adenoviruses and the subsequent analyses.
authors.affiliation: Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA.
authors.forename: Lucy
authors.initials: L
authors.lastname: Pigati
authors.forename: Richard S
authors.initials: RS
authors.lastname: Kang
authors.forename: Heike
authors.initials: H
authors.lastname: Fölsch
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000378:metabolism*; D016764:Cell Polarity; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D016086:Lentivirus / Q000235:genetics; D061985:Madin Darby Canine Kidney Cells; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014779:Virus Replication; D020691:rab GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; C072237:TGOLN2 protein, human; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C085485:RAB13 protein, human; D020691:rab GTP-Binding Proteins
keywords: AP-1B; ARH; Defective virus; Epithelial cell; MDCK; Polarized sorting; Rab13