Using replication defective viruses to analyze membrane trafficking in polarized epithelial cells

Lucy Pigati*, Richard S. Kang, Heike Fölsch

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

2 Scopus citations

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.

Original languageEnglish (US)
Title of host publicationMethods in Cell Biology
PublisherAcademic Press Inc
Pages125-137
Number of pages13
DOIs
StatePublished - 2013

Publication series

NameMethods in Cell Biology
Volume118
ISSN (Print)0091-679X

Keywords

  • AP-1B
  • ARH
  • Defective virus
  • Epithelial cell
  • MDCK
  • Polarized sorting
  • Rab13

ASJC Scopus subject areas

  • Cell Biology

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