title: RNA interference approaches to examine Golgi function in animal cell culture.
abstract: The ability to deplete specific proteins from cells has transformed cell biology. Targeting of gene transcripts using RNA interference has allowed for a highly refined approach to the analysis of gene function that has been applied to all aspects of cell biology. Developments of the technology have reached a point where it is now a relatively trivial task to assess the role of an individual protein in a particular cell function. RNAi also allows for genome-wide screening as a discovery step toward the identification of new components of cellular pathways and machines. The technique has been applied extensively to the analysis of Golgi complex function, leading to significant insight into the biology of this complex organelle. Here, we describe the commonly used options for targeting individual genes for both transient and stable knockdown. We consider the alternative methods for introducing these reagents into cells and outline methods that we and others have used widely for validation of specificity and efficacy of gene targeting.
authors.affiliation: Cell Biology Laboratories, School of Biochemistry, University of Bristol, Bristol, United Kingdom.
authors.forename: Victoria J
authors.initials: VJ
authors.lastname: Miller
authors.forename: Caroline M
authors.initials: CM
authors.lastname: McKinnon
authors.forename: Harry
authors.initials: H
authors.lastname: Mellor
authors.forename: David J
authors.initials: DJ
authors.lastname: Stephens
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006056:Golgi Apparatus / Q000502:physiology*; D000077614:Golgi Matrix Proteins; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D014162:Transfection; D014404:Tubulin; D014776:Virus Cultivation; D033921:Vesicular Transport Proteins
chemical_list: D005700:Galactosyltransferases; D000077614:Golgi Matrix Proteins; D008565:Membrane Proteins; D011506:Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014404:Tubulin; C096163:TFG protein, human; D033921:Vesicular Transport Proteins; C087574:macrogolgin
keywords: COPII; ER exit site; Gene function; Gene suppression; Knockout; RNAi; Transfection; qPCR; shRNA; siRNA