In vitro culture and transposon-mediated genetic modification of chicken primordial germ cells
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Primordial germ cells (PGCs) are the embryonic precursors of the germ cell lineage. Segregation of the chicken germ line from somatic cells occurs very early in embryonic development. By day two of incubation chicken PGCs can be isolated from the circulating blood. The in vitro culture of chicken PGCs has significant potential as a tool for the investigation of germ cell development and as a cell-based system for the production of genetically modified chickens. The isolation, culture and manipulation of migratory chicken PGCs reported previously have not been independently validated. Initial attempts to isolate and culture chicken PGCs by reproducing a published protocol proved difficult. Key components of the published culture medium are by their nature variable, including the use of BRL-conditioned medium and animal sera. The protocol also stated that addition of SCF to the culture medium is essential but did not identify the source of SCF used. Several components of the culture conditions were tested including sources and batches of bovine and chicken sera and the growth factors FGF2 and SCF. Chicken PGCs from wild type and GFPexpressing chicken embryos were cultured and several cell lines established, proliferating for more than 100 days in culture. After seventy days in culture a single chicken PGC cell line was shown to retain the potential to develop into functional sperm. This was demonstrated by injection of the cultured chicken PGCs into early chick embryos, which were hatched and produced offspring derived from the injected chicken PGCs. To understand and produce a more robust system for the isolation and propagation of chicken PGCs three signalling pathways, AKT, MAPK and JAK/STAT, were investigated. When any of these signalling pathways were blocked, using chemical inhibitors, chicken PGC proliferation in vitro was significantly inhibited, showing the pathways to be essential for chicken PGC proliferation. Chicken PGCs were treated with individual components of the standard culture medium, FGF2, SCF, animal sera, BRL-conditioned medium, LIF and IGF, and the activation status of the key signalling pathways was assessed by western blot. Individual components of the culture medium induced activation of the AKT and MAPK pathways but not the JAK/STAT pathway. These data increase our understanding of PGC biology and are the first steps towards the development of a feeder- and serum-free medium for the growth of chicken PGCs. Published methods for the genetic manipulation of chicken PGCs are inefficient. To improve the efficiency of stable transgene integration, transposable element-derived gene transfer vectors were assessed for their ability to transpose into the genome of chicken PGCs. Comparison of Tol2 and piggyBac transposable elements, carrying reporter transgenes, demonstrated that both can be used to genetically-modify chicken cells. The incidence of stable transposition achieved was higher when using the Tol2 transposable element in comparison to the piggyBac element. The genetically-modified chicken PGCs formed functional gametes, demonstrated by injection of genetically modified chicken PGCs into host embryos which were hatched and produced transgenic offspring expressing the reporter gene construct.