title: RNAi screens of lysosomal trafficking.
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.
authors: Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA; Harvard Combined Fellowship in Molecular Pathology, Harvard Medical School, Boston, MA, USA.
authors: Salil
authors: S
authors: Garg
authors: Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA.
authors: Michael B
authors: MB
authors: Brenner
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D015870:Gene Expression; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D021381:Protein Transport; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference
chemical_list: D011506:Proteins; C018589:lysosomal proteins
keywords: Antigen presentation; CD1; Dendritic cell; Lysosome; Lysosome trafficking; MHC II; Macrophage; RNAi; RNAi screen; Vesicle trafficking; shRNA