Investigation of avian germ cell populations

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The goal of this project is to investigate the development of the germ cell lineage during embryogenesis. I seek to understand how germ cells differentiate from a primordial germ cell (PGC), to a gonocyte and finally to a spermatogonial stem cell (SSC). This research will provide insight into the establishment and maintenance of stem cell populations and the inappropriate activation of these cells during the formation of germ cell tumours. I will first establish a PGC culture system to establish cell lines for in vitro and in vivo analysis. These cell lines will be used to define the regulatory regions sufficient to drive germ cell-specific expression. The promoter regions from known avian germ cell factors will be linked to reporter genes and tested for the ability to drive expression in these cells lines. I will subsequently use these validated regulatory regions to generate a germ line-specific transgenic chicken line in order to isolate staged populations of embryonic germ cells. RNA will be purified from three stages of germ cell development and will be profiled by microarray and for microRNAs. These experiments will generate candidate molecules and signalling pathways involved in germ cell development that will be validated and investigated. Candidate molecules will be manipulated by knockdown or overexpression using developed avian vector systems in the PGC lines followed by in ovo transfer. Due to the time delay in generating transgenic chicken lines, potential germ cell regulatory pathways will first be examined using the PGC cultures and subsequently in ovo. This project will take advantage of the unique resources that exist at Roslin Institute, namely, an access to adult chickens and fertile eggs, experience in long term culturing of embryos, an expertise in mammalian embryonic stem cells, and the ability to generate lines of transgenic chickens. This research will have implications for our understanding of the biology of stem cells.

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