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Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy
Published on: December 29, 2017
RNAi screens of lysosomal trafficking
Salil Garg1, Michael B Brenner2
1Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA; Harvard Combined Fellowship in Molecular Pathology, Harvard Medical School, Boston, MA, USA.
Abstract:
Here, we describe the general principles of RNA interference screens to study lysosomal functions in mammalian cells. Lysosomes occupy a central position in the biology of numerous processes such as degradation, microbial killing, and immunological antigen presentation to T cells. Selection of a screening system, conducting pooled versus arrayed screens, and appropriate steps in assay development, validation, and verification of novel gene candidates are all discussed. We focus on our experience in developing an arrayed short hairpin RNA screen to identify novel lysosomal trafficking proteins involved in vesicle and cargo trafficking and illustrate how such a trafficking library can be applied to screens involving lysosomes.
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