Minisymposium XI. New Insights Through Modern Approaches into Gamete Biology and Acquisition of Fertilization Competence
Chair(s): Evans, Janice1, 1 Johns Hopkins, Baltimore, MD
Location: CCQ 205ABC
(MS33) RNAi IN THE OOCYTE: A POWERFUL TOOL TO STUDY GENE FUNCTION IN OOCYTE MATURATION AND FERTILIZATION.
Stein, Paula1, 1 University of Pennsylvania, Philadelphia, PA
ABSTRACT- RNA interference (RNAi) is a conserved post-transcriptional gene silencing mechanism present in most eukaryotes. We have demonstrated that RNAi operates in mouse oocytes and early embryos. We further extended the applicability of RNAi by developing a transgenic RNAi approach to study gene function during oocyte development. The approach utilizes the oocyte-specific Zp3 promoter to drive the expression of a long hairpin double-stranded RNA (dsRNA) that contains sequence complementary to the gene of interest. We validated this approach by targeting Mos and were able to recapitulate the Mos null phenotype. This transgenic RNAi approach has been extended to study the function of other genes involved in oocyte maturation, fertilization and early embryo development. Although RNAi is observed from yeast to humans, mammalian cells possess another pathway that responds to long dsRNA by inducing interferon and expression and activating two dsRNA-dependent enzymes, namely, dsRNA-dependent protein kinase (PKR) and 2′,5′-oligoadenylate synthetase (OAS). PKR activation results in a general inhibition of protein synthesis, while OAS activates RNase L, leading to nonspecific degradation of mRNAs. This antiviral pathway is known as the interferon response. Mouse oocytes and preimplantation embryos seem to lack this response, as potent and specific inhibition of gene expression triggered by long dsRNA is observed in these cells. In order to ascertain whether mouse oocytes can mount an interferon response, we assessed the global pattern of gene expression by microarray analysis in transgenic mouse oocytes exposed to long dsRNA. This analysis, confirmed by real-time PCR and Western blot, shows that PKR, RNase L, and the catalytically active isoforms of OAS are absent in mouse oocytes, while catalytically inactive OAS isoforms, believed to act as dominant negative, are present in large amounts. Transgenic oocytes expressing Mos dsRNA show the same pattern of expression as wild-type oocytes. The microarray analysis also provides invaluable information about the target specificity of RNAi in mouse oocytes and demonstrates the complete absence of off-targeting. We conclude that transgenic RNAi is highly specific in mouse oocytes and therefore is a robust and simple method to study gene function during oocyte development.
KEY WORDS: RNAi, oocyte, interferon, off-targeting