In vitro culture and transposon-mediated genetic modification of chicken primordial germ cells
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Date
30/06/2012Author
Macdonald, Joni
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Abstract
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.
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