Title
Design and delivery of antisense oligonucleotides to block microRNA function in cultured Drosophila and human cells
PubMed ID
18802435
UMMS Affiliation
Department of Biochemistry and Molecular Pharmacology and Howard Hughes Medical Institute
Date
9-20-2008
Document Type
Article
Subjects
Animals; Cells, Cultured; Drosophila; Gene Expression Regulation; Genetic Engineering; Humans; MicroRNAs; *Models, Genetic; Molecular Structure; Oligonucleotides, Antisense; Transfection
Disciplines
Life Sciences | Medicine and Health Sciences
Abstract
MicroRNAs (miRNAs), approximately 22-nt RNAs that mediate post-transcriptional regulation of mRNAs in animals and plants, are a diverse class of regulatory genes whose specific biological functions are largely unknown. Here we detail a protocol to design and introduce into cultured Drosophila and human cells sequence-specific antisense oligonucleotides (ASOs) that block the function of individual miRNAs. Coupled with recent studies that catalog the miRNAs expressed in diverse cultured cells, our method offers a rapid (<1 week) approach to validate miRNA targets and to study the cellular functions of individual human and Drosophila miRNAs. ASO-based inactivation of miRNAs is faster and simpler than comparable genetic or 'sponge'-based approaches, for which extensive recombinant DNA manipulation is required. We present our ASO design principles and an optimized transfection protocol in which transfection efficiency of Drosophila Schneider 2 cells can approach 100%. Our 3'-cholesterol-modified ASOs have enhanced potency, allowing miRNA inhibition for at least 7 d from a single transfection.
Rights and Permissions
Citation: Nat Protoc. 2008;3(10):1537-49. Link to article on publisher's site