RNAi Experiments in Mouse Oocytes and Early Embryos

Abstract

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 provides 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.

RNA interference inCaenorhabditis elegans: Uptake, mechanism, and regulation

“…Control of concentration is critical to maximize the effective dose while simultaneously avoiding non-specific toxicity or off-target effects. The concentration of dsRNA to inject will vary from organism to organism (Kuwabara and Coulson, 2000; Nasevicius and Ekker, 2000; Svoboda and Stein, 2009), from cell type to cell type (Grishok and Mello, 2002; Wang et al 2005), and even from gene target to gene target (Krueger et al 2007). Although in C. elegans silencing signals can spread from the injected cell or tissue, this is not true of other, even closely related species (Winston et al 2007).…”

Sequence-specific inhibition of microRNA via CRISPR/CRISPRi system

“…MicroRNAs (miRNAs), a class of highly conserved non-coding regulatory factors, are present throughout the eukaryotic genome and are predicted to negatively regulate more than half of the protein-coding genes in mammals1. miRNAs are essential to most biological processes, including proliferation, differentiation and apoptosis, and their transcription is tightly controlled1.…”

“…miRNAs are essential to most biological processes, including proliferation, differentiation and apoptosis, and their transcription is tightly controlled1. Aberrant miRNA expression has been associated with numerous diseases.…”