Defining and assaying RNAi in mammalian cells.
The investigation of protein function through the inhibition of activity has been critical to our understanding of many normal and abnormal biological processes. Until recently, functional inhibition in biological systems has been induced using a variety of approaches including small molecule antagonists, antibodies, aptamers, ribozymes, antisense oligonucleotides or transcripts, morpholinos, dominant-negative mutants, and knockout transgenic animals. Although all of these approaches have made substantial advances in our understanding of the function of many proteins, a lack of specificity or restricted applicability has limited their utility. Recently, exploitation of the naturally occurring posttranscriptional gene silencing mechanism triggered by double-stranded RNA (dsRNA), termed RNA interference (RNAi), has gained much favor as an alternative means for analyzing gene function. Aspects of the basic biology of RNAi, its application as a functional genomics tool, and its potential as a therapeutic approach have been extensively reviewed (Hannon and Rossi, 2004; Meister and Tuschl, 2004); however, there has been only limited discussion as to how to design and validate an individual RNAi effector molecule and how to interpret RNAi data overall, particularly with reference to experimentation in mammalian cells. This perspective will aim to consider some of the issues encountered when conducting and interpreting RNAi experiments in mammalian cells.
Huppi
Konrad
K
Gene Silencing Section, Office of Science and Technology Partnerships, Office of the Director, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 37, 3128B, 37 Convent Drive, Bethesda, MD 20892, USA.
Martin
Scott E
SE
Caplen
Natasha J
NJ
D000818:Animals; D002477:Cells / Q000378:metabolism; D005821:Genetic Techniques; D008322:Mammals; D010641:Phenotype; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000235:genetics / Q000378:metabolism; D009626:Terminology as Topic
D011506:Proteins; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex