Design and delivery of antisense oligonucleotides to block microRNA function in cultured Drosophila and human cells
Department of Biochemistry and Molecular Pharmacology and Howard Hughes Medical Institute
Animals; Cells, Cultured; Drosophila; Gene Expression Regulation; Genetic Engineering; Humans; MicroRNAs; *Models, Genetic; Molecular Structure; Oligonucleotides, Antisense; Transfection
Life Sciences | Medicine and Health Sciences
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.
DOI of Published Version
Nat Protoc. 2008;3(10):1537-49. Link to article on publisher's site
Horwich, Michael D. and Zamore, Phillip D., "Design and delivery of antisense oligonucleotides to block microRNA function in cultured Drosophila and human cells" (2008). Open Access Articles. 1947.