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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.08.015 Byline: Paula Stein, Fanyi Zeng, Hua Pan, Richard M. Schultz Keywords: RNAi; dsRNA; Oocyte; Interferon response; Off-targeting Abstract: RNA interference (RNAi) is a conserved eukaryotic mechanism by which double-stranded RNA (dsRNA) triggers the sequence-specific degradation of homologous mRNAs. Recent concerns have arisen in mammalian systems about off-target effects of RNAi, as well as an interferon response. Most mammalian cells respond to long dsRNAs by inducing an antiviral response mediated by interferon that leads to general inhibition of protein synthesis and nonspecific degradation of mRNAs. Moreover, recent reports demonstrate that under certain conditions, short interfering RNAs (siRNAs, 21-25 bp) may activate the interferon system. Mouse oocytes and preimplantation embryos apparently lack this response, as potent and specific inhibition of gene expression triggered by long dsRNA is observed in these cells. In the present study, we analyzed the global pattern of gene expression by microarray analysis in transgenic mouse oocytes expressing long dsRNA and find no evidence of off-targeting. We also report that genes involved in the interferon response pathway are not expressed in mouse oocytes, even after exposure for an extended period of time to long dsRNA. Author Affiliation: Department of Biology, University of Pennsylvania, 415 South University Avenue, Philadelphia, PA 19104-6018, USA Article History: Received 12 July 2005; Revised 11 August 2005; Accepted 12 August 2005

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.03.038 Byline: Fanyi Zeng, Richard M. Schultz Keywords: Microarray; Gene expression; Zygotic gene activation; Preimplantation mouse embryo Abstract: Zygotic gene activation is essential for development beyond the 2-cell stage in the preimplantation mouse embryo. Based on [alpha]-amanitin-sensitive BrUTP incorporation, transcription initiates in the 1-cell embryo and a major reprogramming of gene expression driven by newly expressed genes is prominently observed during the 2-cell stage. Superimposed on genome activation is the development of a transcriptionally repressive state that is mediated at the level of chromatin structure. The identity of the genes that are expressed during the 1- and 2-cell stages, however, is poorly described, as are those genes involved in mediating the transcriptionally repressive state. Using the Affymetrix MOE430 mouse GeneChip set, we characterized the set of [alpha]-amanitin-sensitive genes expressed during the 1- and 2-cell stages, and we used Expression Analysis Systematic Explorer (EASE) and Ingenuity Pathway Analysis (IPA) to identify biological and molecular processes represented by these genes, as well as interactions among them. We find that although the 1-cell embryo is transcriptionally active, we did not detect any transcripts present on the MOE430 GeneChip set to be [alpha]-amanitin-sensitive. Thus, what the BrUTP incorporation represents remains elusive. About 17% of genes expressed in the 2-cell embryo are [alpha]-amanitin-sensitive. EASE analysis reveals that genes involved in ribosome biogenesis and assembly, protein synthesis, RNA metabolism and transcription are over-represented, suggesting that genome activation during 2-cell stage may not be as global and promiscuous as previously proposed. IPA implicated Myc and Hdac1 as candidate genes involved in genome activation and the development of the transcriptionally repressive state, respectively. Author Affiliation: Department of Biology, University of Pennsylvania, 415 South University Avenue, Philadelphia, PA 19104-6018, USA Article History: Received 4 January 2005; Revised 28 March 2005; Accepted 29 March 2005

Studies using low-resolution methods to assess gene expression during preimplantation mouse development indicate that changes in gene expression either precede or occur concomitantly with the major morphological transitions, that is, conversion of the oocyte to totipotent 2-cell blastomeres, compaction, and blastocyst formation. Using microarrays, we characterized global changes in gene expression and used Expression Analysis Systematic Explorer (EASE) to identify biological and molecular processes that accompany and likely underlie these transitions. The analysis confirmed previously described processes or events, but more important, EASE revealed new insights. Response to DNA damage and DNA repair genes are overrepresented in the oocyte compared to 1-cell through blastocyst stages and may reflect the oocyte's response to selective pressures to insure genomic integrity; fertilization results in changes in the transcript profile in the 1-cell embryo that are far greater than previously recognized; and genome activation during 2-cell stage may not be as global and promiscuous as previously proposed, but rather far more selective, with genes involved in transcription and RNA processing being preferentially expressed. These results validate this hypothesis-generating approach by identifying genes involved in critical biological processes that can be the subject of a more traditional hypothesis-driven approach. Keywords: Microarray; Gene expression; Genome activation; Maternal mRNA; Compaction; Blastocyst