Lentivirus-Mediated Antagomir Expression

MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation via hybridisation to mRNAs. miRNA function can be inhibited by the so-called “antagomirs” – anti-sense RNA oligonucleotides complementary to individual miRNAs. Since, in principle, any miRNA can be silenced, antagomirs provide a powerful tool to investigate the function of particular miRNAs. However, conventional methods to deliver antagomirs into cells (e.g. transfection) have been shown to only transiently interfere with endogenous miRNA expression and/or function. In this section, we describe a lentivirus-based system for stable antagomir expression to generate longterm loss-of-function phenotypes for individual miRNAs. Moreover, the described strategy is also suitable for studying the function of individual miRNAs encoded within polycistronic clusters. This chapter provides a collection of protocols to antagonize miRNA function by lentiviral antagomir expression, which can be achieved in a wide range of target cells.