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When the nuclei of differentiated cells are transplanted to enucleated eggs, multipotential embryonic cells can be obtained. These can be made to differentiate into a range of cell types unrelated to the original cells from which the nuclei were taken. This is the basis of a cell replacement strategy by which rejuvenated cells can be derived from an adult cell. These newly-generated cells are of the same genetic constitution as the donor cell, and are therefore suitable for cell replacement therapy without the need for immunosuppression. We aim to identify the molecules and to understand the mechanisms that stabilise cell differentiation during normal development, and that therefore need to be reversed for cell rejuvenation.
Our principal method of analysis involves transplanting multiple nuclei from adult tissues such as the mouse thymus into the growing oocytes of Xenopus. Within two days, or within a few hours for the nuclei of less specialised cells, the transplanted nuclei express genes such as Oct4 and Nanog, which are diagnostic of embryo or stem cells. We analyse the reprogramming of gene expression at several levels. We have found that oocytes have a DNA demethylating activity that reverses differentiation by acting on the promoter of Oct4. We use confocal microscopy to view in real time the binding of defined proteins to somatic cell nuclei as they undergo gene reprogramming.
In related work, we find that single somatic cell nuclei transplanted to enucleated eggs often show epigenetic memory of an active gene state. This seems to be a mechanism for stabilising gene expression in a differentiation pathway, and may account for the decreasing success of nuclear transfers from more differentiated cells.
- Murata K, Kouzarides T, Bannister AJ and Gurdon JB (2010) Histone H3 lysine 4 methylation is associated with the transcriptional reprogramming efficiency of somatic nuclei by oocytes. Epigenetics & Chromatin 3, 4.
- Miyamoto, K, Pasque V, Jullien J and Gurdon JB (2011) Nuclear actin polymerization is required for transcriptional reprogramming of Oct4 by oocytes. Genes & Development 25(9):946-958.
- Simeoni I, Gilchrist, MJ, Garrett N, Armisen J and Gurdon JB (2012) Widespread transcription in an amphibian oocyte relates to its reprogramming activity on transplanted somatic nuclei. Stem Cells & Development 21(2):181-190.
- Pasque V, Jullien J, Miyamoto K, Halley-Stott RP and Gurdon JB (2011) Epigenetic factors influencing resistance to nuclear reprogramming. Trends in Genetics 27(12)516-525.
- Narbonne P, Simpson DE and Gurdon JB (2011) Deficient induction response in a Xenopus nucleocytoplasmic hybrid. PLoS Biology 9(11):e1001197.
- Gurdon, JB (2006) From nuclear transfer to nuclear reprogramming: the reversal of cell differentiation. Ann. Rev. Cell Dev. Biol. 22, 1-22.
- Gurdon JB and Melton DA (2008) Nuclear reprogramming in cells. Science 322, 1811-1815.
- Jullien J, Astrand C, Halley-Stott RP, Garrett N, and Gurdon JB (2010) Characterization of somatic cell nuclear reprogramming by oocytes in which a linker histone is required for pluripotency gene reactivation. PNAS 107, 5483-5488.
- Pasque V, Gillich A, Garrett N, Gurdon JB (2011) Histone variant macroH2A confers resistance to nuclear reprogramming. EMBO J. 6;30(12):2373-87.
- Jullien J, Halley-Stott RP, Miyamoto K, Pasque V and Gurdon JB (2011) Mechanisms of nuclear reprogramming by eggs and oocytes: a deterministic process? Nature Reviews Molecular & Cell Biology, 12, 453-459.