Research topics
Effects of long dsRNA expression in mammalian cells.
We have generated transgenic mice ubiquitously transcribing a long inverted repeat, which should give a rise to dsRNA. These mice do not exhibit any non-specific phenotype except of RNAi effects in the female germline. Characterization of effects of long dsRNA expression in somatic cells of transgenic mice is currently in progress. We are analyzing interferon pathway activation, adenosine deamination of the long dsRNA, RNAi effects, localization of the long dsRNA, etc. Simultaneously, we analyze the same long dsRNA expression system in the cell culture.
Oocyte-to-zygote transition in the mouse.
The zygotic genome activation is the first step in the execution of the genome-encoded program that forms a new organism from a single fertilized cell and it is an essential event in the life of every sexually reproducing organism. The zygotic genome activation is closely associated with formation of pluripotency, i.e the ability of cells to differentiate into any body cell type. Pluripotency is most studied in two artificial cell types, which maintain pluripotency during in vitro culture: embryonic stem cells (ESCs), which are derived from the inner cell mass of the blastocyst, and induced pluripotent stem cells (iPSCs), which form upon reprogramming gene expression in somatic cells with specific pluripotency factors that include transcription factors from the core transcription factor network controlling ESC renewal and pluripotency. A similar network forms in a stepwise manner during the mouse zygotic genome activation, which initiates at the early two-cell stage.
We study reprogramming of oocytes into pluripotent blastomeres of an early embryo (oocyte-to-embryo transition). This model is the natural parallel to the artifical reprogramming of somatic cells into iPSCs. The oocyte-to-embryo transition, however, is distinct. It is a unidirectional transient process executed by cytoplasmic factors, as demonstrated by animal cloning by nuclear transfer. Our primary research interest is in post-transcriptional mechanisms underlying oocyte-to-embryo transition. These mechanisms include control of maternal mRNA stability, small regulatory RNAs in miRNA and RNAi pathways, and production of maternal transcription factors, which will control gene expression in the embryo. Our goal is to understand how control of gene expression creates developmental competence in vivo.
Research of pluripotency is eminent for medicine and biotechnology where pluripotency plays a role in an ever-growing number of applications. Understanding control of the oocyte-to-embryo transition will provide original insights into stem cell biology and will likely contribute to efficient and safe production of pluripotent stem cells, efficient cloning technologies, informative prenatal diagnostics, and understanding of pathology of sterility and developmental defects.
Current Grant Support
2007-2011 EMBO Installation Grant, project 1483, Regulation of mRNA stability during oocyte-to-zygote transition in the mouse.
2009-2013 GAČR, 204/09/0085, Effects of long dsRNA expression in mammalian cells.
2009-2012 KONTAKT-MŠMT, ME09039, Role of post-transcriprtional mechanisms in reprogramming mouse oocytes into pluripotent cells.
2010-2013 GAČR, P305/10/2215, Control of chromatin and pluripotency by microRNAs.
Most important recent papers
(2004-present; names of the lab members underlined)Fedoriw AM, Stein P, Svoboda P, Schultz RM, Bartolomei MS. Transgenic RNAi reveals essential function for CTCF in H19 gene imprinting. Science 303(5655): 238-40, 2004. (Pubmed) (DOI)
Svoboda P, Stein P, Anger M, Bernstein E, Hannon GJ, Schultz RM. RNAi and expression of retrotransposons MuERV-L and IAP in preimplantation mouse embryos. Dev Biol 269(1): 276-85, 2004. (Pubmed) (DOI)
Svoboda P, Stein P, Filipowicz W, Schultz RM. Lack of homologous sequence-specific DNA methylation in response to stable dsRNA expression in mouse oocytes. Nucleic Acids Res 32(12): 3601-6, 2004. (Pubmed) (DOI)
Bultman SJ, Gebuhr TC, Pan H, Svoboda P, Schultz RM, Magnuson T. Maternal BRG1 regulates zygotic genome activation in the mouse. Genes Dev 20(13): 1744-54, 2006. (Pubmed) (DOI)
Puschendorf M, Stein P, Oakeley EJ, Schultz RM, Peters AH, Svoboda P. Abundant transcripts from retrotransposons are unstable in fully grown mouse oocytes. Biochem Biophys Res Commun 347(1): 36-43, 2006. (Pubmed) (DOI)
Schmitter D, Filkowski J, Sewer A, Pillai RS, Oakeley EJ, Zavolan M, Svoboda P, Filipowicz W. Effects of Dicer and Argonaute down-regulation on mRNA levels in human HEK293 cells. Nucleic Acids Res 34(17): 4801-15, 2006. (Pubmed) (DOI)
Sinkkonen L, Hugenschmidt T, Berninger P, Gaidatzis D, Mohn F, Artus-Revel CG, Zavolan M, Svoboda P, Filipowicz W. MicroRNAs control de novo DNA methylation through regulation of transcriptional repressors in mouse embryonic stem cells. Nat Struct Mol Biol 15(3): 259-67, 2008. (Pubmed) (DOI)
Flemr M, Ma J, Schultz RM, Svoboda P. P-Body Loss Is Concomitant with Formation of a Messenger RNA Storage Domain in Mouse Oocytes. Biol Reprod. 2010 Jan 14. [Epub ahead of print] (Pubmed) (DOI)
Ma J, Flemr M, Stein P, Berninger P, Malik R, Zavolan M, Svoboda P, Schultz RM. MicroRNA activity is suppressed in mouse oocytes. Curr Biol 20(3): 265-70, 2010. (Pubmed) (DOI)
Sarnova L, Malik R, Sedlacek R, Svoboda P. Shortcomings of short hairpin RNA-based transgenic RNA interference in mouse oocytes. J Negat Results Biomed 9: 8, 2010. (Pubmed) (DOI)
Sinkkonen L, Hugenschmidt T, Filipowicz W, Svoboda P. Dicer is associated with ribosomal DNA chromatin in mammalian cells. PLoS One 5(8): e12175, 2010. (Pubmed) (DOI)
Reviews
Stein P, Svoboda P. Guide to RNAi in mouse oocytes and preimplantation embryos. Cold Spring Harbor Press August 2003 (book chapter).
Svoboda P. Long dsRNA and silent genes strike back:RNAi in mouse oocytes and early embryos. Cytogenet Genome Res 105(2-4): 422-34, 2004. (Pubmed) (DOI)
Horman SR, Svoboda P, Prak ET. The potential regulation of l1 mobility by RNA interference. J Biomed Biotechnol 2006(1): 32713, 2006. (Pubmed) (DOI)
Svoboda P, Di Cara A. Hairpin RNA: a secondary structure of primary importance. Cell Mol Life Sci 63(7-8): 901-8, 2006. (Pubmed) (DOI)
Svoboda P. Off-targeting and other non-specific effects of RNAi experiments in mammalian cells. Curr Opin Mol Ther 9(3): 248-57, 2007. (Pubmed)
Grosshans H, Svoboda P. miRNA, piRNA, siRNA -- Kleine Wiener Ribonukleinsauren. Bioessays 29(9): 940-3, 2007. (Pubmed) (DOI)
Svoboda P. RNA silencing in mammalian oocytes and early embryos. Curr Top Microbiol Immunol 320: 225-56, 2008. (Pubmed)
Kutter C, Svoboda P. miRNA, siRNA, piRNA: Knowns of the unknown. RNA Biol 5(4): 181-8, 2008. (Pubmed)
Svoboda P. MicroRNAs in stem cells. Controlling Pluripotency and Differentiation. Bioforum Europe 11/2008: 28-30, 2008.
Svoboda P, Stein P. RNAi experiments in mouse oocytes and early embryos. CSH Protoc 2009(1): pdb.top56, 2009. (Pubmed) (DOI)
Svoboda P. Why mouse oocytes and early embryos ignore miRNAs? RNA Biol 7(5): 559-63, 2010. (Pubmed)
Flemr M, Svoboda P. Ribonucleoprotein localization in mouse oocytes. Methods 53(2): 136-41, 2011. (Pubmed) (DOI)
Svoboda P, Flemr M. The role of miRNAs and endogenous siRNAs in maternal-to-zygotic reprogramming and the establishment of pluripotency. EMBO Rep 11(8): 590-7, 2010. (Pubmed) (DOI)
Teaching, lectures, public presentations
MB150P85 - Epigenetics - Faculty of Science, Charles University, Prague; semestral course in Czech.
Co-organizer of Advances in Molecular Biology and Genetics