RNAi Experiments in Mouse Oocytes and Early Embryos Petr Svoboda 1,3 and Paula Stein 2,3 1 Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic 2 Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA INTRODUCTION The discovery of RNA interference (RNAi) in 1998 ushered in a new era in biology. RNAi currently serves as a favorite approach for inhibition of gene function in many areas of research. This article pro- vides a brief review of RNAi and discussion of the benefits and drawbacks of using long double- stranded RNA (dsRNA) in mammalian oocytes and early embryos. We also provide an introduction to protocols for RNAi experiments in mouse, including preparation and microinjection of dsRNA into mouse oocytes and early embryos, and preparation and testing of constructs for transgenic RNAi based on long hairpin RNA expression. RELATED INFORMATION Protocols for Preparation of dsRNA for Microinjection Experiments in Mouse (Svoboda 2009a), Microinjection of dsRNA into Fully-Grown Mouse Oocytes (Stein 2009a), Microinjection of dsRNA into Mouse One-Cell Embryos (Stein 2009b), Cloning a Transgene for Transgenic RNAi in Mouse Oocytes (Svoboda 2009b), and Microinjection of Plasmids into Meiotically Incompetent Oocytes (Stein 2009c) are available. This protocol set provides an updated version of protocols previously pub- lished as Preparation of dsRNA Molecules for RNAi in Mouse Oocytes and Early Embryos (Stein and Svoboda 2006a), Collection of Mouse Oocytes for RNAi (Stein and Svoboda 2006b), Collection of Early Mouse Embryos for RNAi (Stein and Svoboda 2006c), Microinjection of dsRNA into Mouse Oocytes and Early Embryos (Stein and Svoboda 2006d), Synthesis of dsRNA for RNAi in Drosophila: Plasmid Template Method (Carthew 2006), and RNA Isolation and RT-PCR from dsRNA-Treated Mouse Oocytes and Early Embryos (Stein and Svoboda 2006e). Articles describing Choosing the Sequence of dsRNA for RNAi in Mouse (Svoboda 2009c) and Cloning and Sequencing an Inverted Repeat (Svoboda 2009d) are also available. BACKGROUND RNAi is a common experimental approach used to induce sequence-specific gene silencing in mam- malian cells. RNAi belongs to a group of so-called RNA silencing pathways, which employ small RNAs as sequence-specific guides for inhibition of gene expression. In mammals, two closely related post-transcriptional RNA silencing pathways, RNAi and microRNA (miRNA), regulate gene expres- sion by inducing degradation and/or translational repression of target mRNAs. These pathways are initiated by different dsRNA molecules that are processed by Dicer, an RNase III endonuclease, into 21- to 22-nucleotide (nt)-long RNA molecules that serve as sequence-specific guides for silencing (Fig. 1; for reviews, see Sontheimer 2005; Zamore and Haley 2005). These small RNAs are termed siRNAs (for small interfering RNAs produced by Dicer-mediated cleavage of long dsRNA substrates) © 2009 Cold Spring Harbor Laboratory Press 1 Vol. 4, Issue 1, January 2009 3 Corresponding authors (svobodap@img.cas.cz; steinpau@sas.upenn.edu) Cite as: Cold Spring Harb. Protoc.; 2009; doi:10.1101/pdb.top56 www.cshprotocols.org Topic Introduction