Published online 19 November 2007 Nucleic Acids Research, 2007, Vol. 35, No. 22 e149 doi:10.1093/nar/gkm971 Lentivirus-mediated antagomir expression for specific inhibition of miRNA function Michaela Scherr*, Letizia Venturini, Karin Battmer, Michael Schaller-Schoenitz, Daniel Schaefer, Iris Dallmann, Arnold Ganser and Matthias Eder Department of Hematology, Hemostasis, and Oncology, Hannover Medical School, Hannover, Germany Received July 22, 2007; Revised September 20, 2007; Accepted October 18, 2007 ABSTRACT Micro RNAs (miRNA) regulate gene expression by hybridization and recruitment of multi-protein com- plexes to complementary mRNA target sequences. miRNA function can transiently be antagonized by antagomirs—chemically modified oligonucleotides complementary to individual miRNAs. Here, we des- cribe the induction of stable loss-of-function pheno- types for specific miRNAs by lentivirus-mediated antagomir expression. Lentivirally expressed antag- omirs are transcribed from a H1-promoter located within the lentiviral 3’LTR and were directed against miRNAs encoded on the polycistronic miR17-92 transcript. Functional silencing of miR-18a, miR- 19b and miR-20a by the corresponding antagomirs specifically relieves miRNA-mediated reporter gene repression. Inhibition of miRNA function correlates to reduction of ‘miRNA’ amplification by miRNA- specific quantitative RT-PCR. Furthermore, protein expression of E2F-1, a known miR-20 target, is enhanced by lentivirally expressed anti-miR-20 antagomirs in a dose-dependent manner, whereas over-expression of miR-20a reduces E2F-1 levels. Finally, combined over-expression of specific miRNAs and antagomirs reveals individual and complementary functions of miR-18a and miR-20a and demonstrates specific miRNA impact on cell proliferation in a cell culture model. INTRODUCTION MicroRNAs (miRNA) are small non-coding RNAs, which are part of an evolutionarily highly conserved intracellular mechanism to regulate gene expression in a sequence-speciﬁc manner (1). miRNAs were initially discovered by analysis of mutations causing developmen- tal defects in Caenorhabditis elegans (2), but recent work also demonstrates altered miRNA expression in human cancer including leukemia (3–7). Accordingly, we recently demonstrated BCR-ABL and c-MYC-dependent aberrant expression of the polycistronic miR17-92 miRNA cluster in early phase of chronic myeloid leukemia (CML) (7). So far, the function of and the targets regulated by individual miRNAs, in particular of those encoded on polycistronic transcripts, are largely unknown. Analysis of miRNA function, for example in the context of clonal evolution, requires methods to generate stable gain- and loss-of-function phenotypes for individual miRNAs and subsequent identiﬁcation and isolation of modiﬁed cells. Stable gain-of-function can be achieved by over- expression of individual miRNAs upon retro- or lentiviral transfer of suitable miRNA-expression cassettes (8–10). In contrast, no method to induce stable loss-of-function phenotypes for individual miRNAs has yet been reported. So far, chemically modiﬁed antisense oligonucleotides complementary to speciﬁc miRNAs, so-called ‘antago- mirs’, have been shown to transiently interfere with miRNA function in cell culture reporter assays and in mice (11–13). However, tracking and isolation of cells with reduced miRNA function has not yet been achieved. Here we describe the generation of stable gain- and loss- of-function phenotypes for individual miRNAs by lenti- viral transfer of antagomir and miRNA expression cassettes, respectively. We demonstrate speciﬁc inhibition of miRNA function in reporter assays by lentivirally encoded anti-miRNA ‘antagomirs’ (hereafter referred to as antagomir). This inhibition correlates to reduced ‘miRNA’ ampliﬁcation by miRNA-speciﬁc quantitative RT-PCR (miR-qRT-PCR). Using cell lines transduced with anti-miR-20a antagomirs and sorted according to their EGFP (green ﬂuorescence protein) ﬂuorescence intensity, we demonstrate that protein expression of E2F-1, a known target of miR-20a, correlates inversely with the miR-20a level as determined by miR-qRT-PCR. Finally, combined over-expression of individual miRNAs and antagomirs deﬁnes complementary functions of miR- 17-92-encoded miRNAs with a negative and positive *To whom correspondence should be addressed. Tel: +49 511 532 9207; Fax: +49 511 532 9242; Email: m.scherr@t-online.de Correspondence may also be addressed to Matthias Eder. Tel: +49 511 532 9207; Fax: +49 511 532 9242; Email: eder.matthias@mh-hannover.de 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.