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Content archived on 2024-05-29

Identification and functional analysis of genes from mouse primordial germ cells that are implicated in the epigenetic reprogramming of the genome

Objective

Development from a totipotent zygote into an adult occurs essentially without changes in DNA sequences but involves epigenetic mechanisms. This includes modifications of histones and DNA such as methylation, which regulate expression of specific set of genes present in all cells. Similar mechanisms are also involved during differentiation of pluri potent stem cells. These modifications are however reversible, for example, when somatic nuclei re-acquire totipotency when transplanted into oocytes. There is also an extensive erasure of most of the pre-existing epigenetic information in primordial germ cells (PGCs) immediately after their entry into the developing gonads, which provides a unique opportunity to discover the mechanism of epigenetic reprogramming of the genome. PGCs until about E10.5 possess modifications of the DNA that are similar to those in somatic cells, such as DNA methylation of specific regulatory elements, as well as the inactive X chromosome. However, when PGCs enter into the developing gonads, this information is erased, rapidly and substantially by E12.5.

This process is prerequisite for subsequent totipotency of the zygote. I propose to identify and study the key genes involved in the erasure of epigenetic information from PGCs. PGCs will be isolated from genital ridges at E10 and from E11.5. Key candidate genes that are activated between E10.0 and E11.5 will be identified using Representational Difference Analysis (RDA) and micro-array analysis. Subsequently, verification of their expression patterns will be carried out by in situ hybridisation and real time RT-PCR on PGCs isolated from E9.5 to E12.5. Functional analysis of the candidates will involve cell-based assays in vitro, and siRNA in ES cells. These modified ES cells will be introduced into host blastocysts, and PGCs from the resulting fetuses will be analysed for their effects on epigenetic reprogramming. I will also examine if any of these factors are inherited in oocytes to determine.

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Topic(s)

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Call for proposal

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FP6-2002-MOBILITY-3
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Funding Scheme

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EIF - Marie Curie actions-Intra-European Fellowships

Coordinator

THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE
EU contribution
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Total cost

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