In vitro culture and transposon-mediated genetic modification of chicken primordial germ cells

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

Characterisation and Germline Transmission of Cultured Avian Primordial Germ Cells

Background: Avian primordial germ cells (PGCs) have significant potential to be used as a cell-based system for the study and preservation of avian germplasm, and the genetic modification of the avian genome. It was previously reported that PGCs from chicken embryos can be propagated in culture and contribute to the germ cell lineage of host birds. Principal Findings: We confirm these results by demonstrating that PGCs from a different layer breed of chickens can be propagated for extended periods in vitro. We demonstrate that intracellular signalling through PI3K and MEK is necessary for PGC growth. We carried out an initial characterisation of these cells. We find that cultured PGCs contain large lipid vacuoles, are glycogen rich, and express the stem cell marker, SSEA-1. These cells also express the germ cell-specific proteins CVH and CDH. Unexpectedly, using RT-PCR we show that cultured PGCs express the pluripotency genes c-Myc, cKlf4, cPouV, cSox2, and cNanog. Finally, we demonstrate that the cultured PGCs will migrate to and colonise the forming gonad of host embryos. Male PGCs will colonise the female gonad and enter meiosis, but are lost from the gonad during sexual development. In male hosts, cultured PGCs form functional gametes as demonstrated by the generation of viable offspring. Conclusions: The establishment of in vitro cultures of germline competent avian PGCs offers a unique system for the study of early germ cell differentiation and also a comparative system for mammalian germ cell development. Primary PGC lines will form the basis of an alternative technique for the preservation of avian germplasm and will be a valuable tool fo

Non-commutative Pieri operators on posets

We consider graded representations of the algebra NC of noncommutative symmetric functions on the Z-linear span of a graded poset P. The matrix coefficients of such a representation give a Hopf morphism from a Hopf algebra HP generated by the intervals of P to the Hopf algebra of quasi-symmetric functions. This provides a unified construction of quasi-symmetric generating functions from different branches of algebraic combinatorics, and this construction is useful for transferring techniques and ideas between these branches. In particular we show that the (Hopf) algebra of Billera and Liu related to Eulerian posets is dual to the peak (Hopf) algebra of Stembridge related to enriched P-partitions, and connect this to the combinatorics of the Schubert calculus for isotropic flag manifolds.Comment: LaTeX 2e, 22 pages Minor corrections, updated references. Complete and final version, to appear in issue of J. Combin. Th. Ser. A dedicated to G.-C. Rot

Hopf Algebras of Graphs

We define graded Hopf algebras with bases labeled by various types of graphs and hypergraphs, provided with natural embeddings into an algebra of polynomials in infinitely many variables. These algebras are graded by the number of edges and can be considered as generalizations of symmetric or quasi-symmetric functions

Hopf algebras and Markov chains: Two examples and a theory

The operation of squaring (coproduct followed by product) in a combinatorial Hopf algebra is shown to induce a Markov chain in natural bases. Chains constructed in this way include widely studied methods of card shuffling, a natural "rock-breaking" process, and Markov chains on simplicial complexes. Many of these chains can be explictly diagonalized using the primitive elements of the algebra and the combinatorics of the free Lie algebra. For card shuffling, this gives an explicit description of the eigenvectors. For rock-breaking, an explicit description of the quasi-stationary distribution and sharp rates to absorption follow.Comment: 51 pages, 17 figures. (Typographical errors corrected. Further fixes will only appear on the version on Amy Pang's website, the arXiv version will not be updated.

The oncogene Gankyrin is expressed in testicular cancer and contributes to cisplatin sensitivity in embryonal carcinoma cells

BACKGROUND: Testicular germ cell cancer (TGCC) develops from pre-malignant germ neoplasia in situ (GCNIS) cells. GCNIS originates from fetal gonocytes (POU5F1+/MAGE-A4-), which fail to differentiate to pre-spermatogonia (POU5F1-/MAGE-A4+) and undergo malignant transformation. Gankyrin is an oncogene which has been shown to prevent POU5F1 degradation and specifically interact with MAGE-A4 in hepatocellular carcinoma (HCC) cells. We aimed to investigate the role of Gankyrin in progression from gonocyte to pre-invasive GCNIS and subsequent invasive TGCC. METHODS: We determined Gankyrin expression in human fetal testicular tissue (gestational weeks 9-20; n = 38), human adult testicular tissue with active spermatogenesis (n = 9), human testicular tissue with germ cell maturation delay (n = 4), testicular tissue from patients with pre-invasive GCNIS (n = 6), and invasive TGCC including seminoma (n = 6) and teratoma (n = 7). Functional analysis was performed in-vitro by siRNA knock-down of Gankyrin in the NTera2 cells (derived from embryonal carcinoma). RESULTS: Germ cell expression of Gankyrin was restricted to a sub-population of prespermatogonia in human fetal testes. Nuclear Gankyrin was also expressed in GCNIS cells of childhood and adult pre-invasive TGCC patients, and in GCNIS from seminoma and non-seminoma patients. Cytoplasmic expression was observed in seminoma tumour cell