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MicroRNAs (miRNAs) are small, single stranded RNA molecules ranging between 19-22nt in length, that are mainly responsible for maintaining cellular homoeostasis by epigenetically regulating the mRNA translation. Each individual miRNA possesses a signature seed sequence spanning between 2-8nt in length. The seed sequence specifically binds to the untranslated regions (UTR) occurring at the either end of functional mRNA molecules based on Watson and Crick complementarity which decides the translational fate of target mRNAs into functionally active proteins. Amongst the many miRNAs discovered, miRNA-155 (miR-155) is one of the most studied miRNAs due to its diverse functional roles in several biological processes. For example, miR-155 expression is reported to be essential in maintaining germinal centers as well as in biogenesis of immunologically functional B cell lineages. Suppression of miR-155 causes immunological impairment in B cells. Interestingly, miR-155 is found to be over-expressing in several human lymphomas including those caused by oncogenic viruses. Some of the examples include diffuse large B cell lymphoma, chronic lymphocytic leukemia, as well as in Epstein-Barr virus (EBV)-induced B cell transformation. All mis-express the miR-155 pathway in human cancers. Furthermore, the occurrence of viral orthologues of miR-155 in oncogenic viruses for e.g., kshv-miR-k12 in Kaposi sarcoma human virus (KSHV), along with their induced expression in human cancers highlights the functional involvement of this molecule in oncogenesis. A similar condition of neoplastic transformation is observed in lymphoid organs of Galliformes which is mainly caused by different avian oncogenic viruses such as Marek's disease virus (MDV), avian leukosis virus (ALV) and reticuloendotheliosis virus (REV), that utilize the gga-miR-155 pathway. MDV, possesses a functional orthologue of gga-miR-155 known as the mdv1-miR-M4 that is upregulated and is proven to be essential for neoplastic transformation. The main goal of my PhD project is to perform an in-depth investigation of the underlying pathways regulated by gga-miR-155 in chicken tumour cell lines. I have employed CRISPR/Cas9 based genome editing for genetic ablation of gga-miR-155 in several chicken cell lines, including DF-1 fibroblast cells, HP45 (ALV subtype-A-induced B cell lymphoma cell line), AVOL-1 (REV-T strain transformed T cell lymphoma cell line) and chicken primordial germ cells (chPGCs). First, I have generated purified single cell clones of DF-1 cell line with ablated gga-miR-155 locus using CRISPR/Cas9 and demonstrated the feasibility and efficiency of CRISPR/Cas9 gene editing in a chicken cell line. I utilized the same gRNAs for genetic editing of gga-miR-155 locus in the ALV-transformed. B-cell lymphoma-derived HP45 cell line, as well as in the primary chicken primordial germ cells (PGC). Successful generation of gga-miR-155 deleted clones of HP45 and chicken PGCs demonstrated that expression of gga-miR-155 is not essential for the maintenance and proliferation of these cell types. Furthermore, gga-miR-155 deletion in chicken PGCs will allow the generation of gga-miR-155 knockout transgenic chickens in the future to study and understand effects of viral replication as well as oncogenesis in in vivo environment lacking gga-miR-155 expression. Derivation of purified single cell clones from genetically edited HP45 cell line allowed me to explore the functionality of gga-miR-155 in the following aspects - 1) In understanding involvement of gga-miR-155 on the biology, morphology, viability, and proliferation of the HP45 cell line. 2) In exploring differentially regulated genes (DRGs) using genome-wide high-throughput analysis: RNA sequencing as well as mass spectrometry and in analyzing effect of genetic modification on downstream genetic pathways by a bio-informatic approach. 3) Using the DRGs for characterizing new targets of gga-miR-155 by luciferase reporter assays. 4) And, in understanding the impact of gga-miR-155 ablation on the ALV replication which was analyzed by ELISA based ALV quantification. ALV, an alpha retrovirus of the family Retroviridae, induces activation of host genes by insertional mutagenesis. However, the specificity and selectivity of the insertion sites in insertional upregulation of host genes is not known. A duplicated long terminal repeat region (LTR) on either end of the virus, with strong promoter-enhancer function, is responsible for inducing the host gene expression. In the HP45 cell line, the ALV subgroup-A upregulates the host genes by integration of its LTR. According to earlier reports, ALV LTR showed integration and upregulation of MYC and BIC (gga-miR-155 host gene) in addition to other genes such as MYB and TERT that are oncogenic in nature. I confirmed that the HP45 cell line also showed induced expression of both MYC and BIC. Further, I have mapped for new integration sites by the Targeted Locus Amplification (TLA) (a high-throughput technique) in the same cell line. As a result, 8 new genes are identified, of which I successfully characterized 5 which showed successful upregulation.

Primordial germ cells (PGCs) are germline competent cells which form the functional gametes of the animal. The potential usage of avian PGCs in producing genetically modified birds has driven research in the derivation, culturing, and genetic manipulation of PGCs. In chicken blastoderm, approximately 50 PGCs are present which proliferate in both male and female embryos until stage HH34 (day 8) and subsequently follow different differentiation pathways in male and female gonads. I investigated the hypothesis that chicken migratory stage PGCs are not initially determined to an oocyte or spermatogonial fate. To understand the differences in genetic mechanisms between male and female chicken PGCs, I studied the RNA transcriptome of PGCs from chicken. Analysis of RNA-Seq data of chicken PGCs reveals transcriptome divergence between the male and female cells and identified 150 differentially expressed genes (DEGs). The cultured female PGCs showed higher expression of cell adhesion genes like NCAM2 and PCDH9, and SMAD7B than male PGCs and also showed that dosage compensation is not maintained throughout the Z sex chromosome. To identify novel germ cell and stem cell factors in avian PGCs, I compared the transcriptome of chicken PGCs with immortalized chicken cell lines. As a result, a set of genes were identified which are specific to germ cells including DAZL, DDX4, DDX43, PNLDC1, DMRT1, DMRTB1, and FKBP6. This analysis also helped to identify a suite of pluripotency genes expressed in PGCs: NANOG, OCT4, LIN28, SOX3, GNOT1, TGIF2, PRDM14 and many others. Furthermore, a cross-species transcriptome comparison between in vitro cultured chicken and goose PGC transcriptomes revealed that the expression of these sets of germ cell-specific genes and pluripotent genes expression is conserved in PGCs from these two avian species. This study also revealed the contrasting gene regulatory networks involved in the selfrenewal are active in chicken and goose PGCs. Chicken PGCs exhibit expression of both Activin and BMP signalling pathway genes whereas BMP signalling pathway genes are active in goose PGCs. PRDM14 belongs to the family of the transcription factors containing a conserved N-terminal SET regulatory domain. In mouse, Prdm14 gene expression is limited to the pluripotent cells and essential for the development of the germ cell lineage. In chicken, the PRDM14 knockout embryos do not form a primitive streak. I characterized germ cell development in PRDM14 null chicken embryos and found that PRDM14 has a crucial role in the survival and maintenance of germ cells. Extending my transcriptome analysis to wild-type and PRDM14 null embryos identified DEGs and regulatory pathways possibly responsible for the gastrulation phenotype in the null embryos.

The mononuclear phagocyte system (MPS), which is a heterogenous family of functionally related cells, includes myeloid progenitors, blood monocytes, resident tissue macrophages, bone osteoclasts and conventional dendritic cells. In mammals, macrophage colony stimulating factor (M-CSF or CSF1) promote differentiation, proliferation and survival of myeloid progenitor cells into mononuclear phagocyte lineage cells by binding and signalling activity through a surface receptor (CSF1R). Interleukin-34 or IL34 is alternative growth factor which also signals via CSF1R. CSF1, IL34 and the shared receptor CSF1R was shown to be conserved in birds, but their functions have not been studied in detail. The primary aim of this project is to study the role of CSF1R signalling in avian macrophage biology using three different approaches. The first approach involved the identification of chicken CSF1R specific kinase inhibitors, from a set of candidate mammalian CSF1R. Candidate CSF1R inhibitors were screened based on cell viability assay using IL-3 dependent pro B cell line Ba/F3 ectopically expressing chicken CSF1R and chicken bone marrow-derived macrophages (BMDM). To support these studies, biologically active, endotoxin-free recombinant chicken CSF1 protein was produced and refolded from inclusion bodies using a bacterial system. Out of 10 potential CSF1R inhibitors screened, 6 inhibitors TIA086, TIA02-052, TIA02-054, TIA02-076, KUL01-123 and KUL02- 016 were potent and selective for chicken CSF1R, whilst having no effect on growth in IL-3. Two inhibitors TIA02-054 and TIA02-076 were specific for the chicken CSF1R kinase compared to their actions on human CSF1R expressed in the same cells. The chicken CSF1R kinase inhibitors also effectively blocked CSF1-induced survival of primary BMDMs. BMDM survival was reduced even in the absence of exogenous CSF1 indicating a growth factor independent, autocrine CSF1/CSF1R signalling function in chicken macrophages. The second approach to study CSF1 biology in the development of chicken MPS involved use of a novel neutralising monoclonal antibody to chicken CSF1 (ROS-AV183) that targets and blocks chicken CSF1R signalling activity. In order to test the activity of anti-ChCSF1 mAb on chicken macrophages both in vitro and in vivo, both anti-ChCSF1 mAb and Isotype control mAb reagents were purified from hybridoma culture by affinity chromatography and characterized further for purity, size by SDS PAGE and CSF1R signaling blocking activity by BaF3/ChCSF1R cell viability assay. Anti-ChCSF1 mAb completely inhibited survival of primary chicken macrophages, irrespective of the presence or absence of CSF1, supporting the earlier finding regarding the autocrine CSF1 signalling behaviour of chicken macrophages. To determine the impact of anti-ChCSF1 mAb on postnatal birds in vivo, transgenic CSF1R-eGFP reporter birds were injected with antibody for four consecutive days. Anti-ChCSF1 mAb had no effect on the average growth rate, the relative weight gain or the normal development of hatchling birds. Anti-ChCSF1 mAb had no detectable effect on circulating CSF1 levels on the day of hatch or a week after treatment. Anti-ChCSF1 mAb significantly reduced CSF1R-eGFP transgene positive macrophages in bursa of Fabricius and caecal tonsil tissue, but not in spleen tissue. In bursa of Fabricius tissue, follicle associated epithelium (FAE) cell's proliferation and survival was altered post treatment. In caecal tonsil anti-ChCSF1 mAb substantially reduced B lymphocytes; this depletion was also evident in the circulation and spleen tissue. Tissue resident MHC-II+ macrophages in spleen were effectively depleted, validating CSF1 dependency of tissue resident macrophages. In liver tissue, anti-ChCSF1 mAb treatment completely ablated Kupffer cell population. In bones anti-ChCSF1 mAb treatment depleted osteoclasts number. MicroCT scan analysis of bone femur architecture revealed significant reduction in the % bone volume and trabecular number, with a corresponding increase in the trabecular separation post anti-ChCSF1 mAb treatment of hatchling birds. In overview, the analysis indicated that CSF1 is required for post-hatch development of the MPS in birds and suggest trophic roles for CSF1-dependent macrophages in B cell development. The third approach involved deletion of CSF1R in the chicken genome using CRISPR Cas9 editing in chicken primordial germ cells (PGCs). Out of the several guide RNAs (gRNAs) designed targeting different regions of CSF1R loci, gRNAs targeting exon 1 and 10 (encoding transmembrane domain of the receptor) were functionally validated for mutation. Guide RNAs targeting exon 1 and transmembrane domain region were effective in mutating receptor CSF1R in cultured PGCs with targeting efficiency of around 35% and 100% respectively. Transplantation of PGCs with biallelic deleted transmembrane domain region of CSF1R into germ cell deficient chicken embryos gave rise to one founder female G0 bird containing edited donor PGCs. Breeding of this chicken upon sexual maturation with transgenic CSF1R-eGFP male established 30 CSF1R heterozygous G1 birds containing CSF1R edited donor PGCs (39% germline efficiency). CSF1R heterozygous G1 birds had no obvious phenotypes compared to wild type hatch mates throughout the development of embryos and in adults. Furthermore, CSF1R homozygous mutant embryos (G2) were generated by breeding CSF1R heterozygous G1 chickens (26% germline efficiency). Analysis of 8-day old CSF1R homozygous mutant embryos revealed deficiency in the expression of CSF1R protein in mononuclear phagocyte population. Hence, there was successful transmission of CSF1R knockout allele in G1 and G2 progeny. Analysis of the phenotype of the homozygous CSF1R mutant birds is ongoing. The novel tools characterized in this project, anti-ChCSF1 antibody, chicken CSF1R kinase domain inhibitors and CSF1R-deficient transgenic chicken line will enable further detailed studies of the role of macrophages in chicken immunity and development.

More than 1500 proteins have been identified as centrosome or cilia proteins, however it is still unknown how these proteins form networks to control functions such as centrosome orientation, maturation and ciliogenesis. TALPID3 is a centrosomal protein that plays a role in centrosome orientation and migration, ciliogenesis and Hedgehog pathway signal transduction. Although several proteins that interact with TALPID3 have been identified, the TALPID3 protein interacting network has not been established. Loss of TALPID3 leads to a loss of ciliogenesis, as seen in the embryonic lethal talpid3 chicken. Unlike the talpid3 chicken however, human ciliopathy patients with mutations in KIAA0586 (human TALPID3 orthologue) have a range of defects that often do not result in lethality. Human KIAA0586 was modelled in the chicken, in order to understand the functional domains that are affected by mutations that are predicted to be hypomorphic. A construct containing human KIAA0586 was able to rescue Hedgehog-dependent expression patterns, when electroporated into the developing neural tube of talpid3 chicken embryos. An attempt to introduce a fluorescent tag at the endogenous TALPID3 locus to look at specific subcellular localisation, proved unsuccessful; however a super resolution imaging approach confirmed localisation of TALPID3 to the centrosome. Structured Illumination Microscopy showed that TALPID3 localises to the distal end of centrioles with more TALPID3 on one centriole than the other centriole, but no specific regions of localisation elsewhere in the cell. A proteomic approach on isolated centrosomes was undertaken to investigate the TALPID3 protein network. Proteomic studies in the centrosome have previously been carried out in KE37 cells, however in order to establish alternative cell lines, specifically chicken Primordial Germ Cells (PGCs), as models for studying the centrosome and cilia, this study was completed in human Jurkat cells and chicken PGCs. PGCs can be derived from the talpid3 flock, therefore by including chicken PGCs the aim was to construct the centrosomal proteome of talpid3 vs wildtype centrosomes from talpid3 PGCs. Through this study, I have shown that Jurkat cells have a low frequency of ciliogenesis with modifications of the centrioles that correlate with their low ciliogenesis frequency; however PGCS are capable of forming cilia and have modifications of the centrioles that correlate with their ability to form cilia, highlighting PGCs as a suitable cell model for carrying out centrosome and cilia studies. A mass spectrometry screen on centrosomes isolated from Jurkat cells and PGCs failed to identify TALPID3; however, the screen identified CCDC77 and CCDC127 as novel centrosome proteins, which was confirmed by immunofluorescence. CRISPR/Cas9 editing, demonstrated that mutations in CCDC127 resulted in a significant reduction of ciliogenesis and altered centrosome protein localisation patterns in RPE1 cells. Isolation of centrosomes from PGCs did not prove effective for constructing the proteome of talpid3, therefore an alternative approach was taken using protein extracted from whole cell lysate of talpid3 and wildtype PGCs. A quantitative proteomic study was undertaken in talpid3 PGCs with the aim of understanding how protein networks are altered in talpid3. Following a Tandem Mass Tag (TMT) mass spectrometry approach, pathway analysis in Ingenuity Pathway Analysis software (IPA) identified down-regulation of protein pathways linked to regulation of the actin cytoskeleton and showed an overall change to protein pathways associated with cholesterol biosynthesis. Immunofluorescence in talpid3 embryo sections was used to examine changes to proteins involved in actin regulation, including F-actin, Profilin, Cofilin, Twinfilin and RhoC. The main findings of this thesis include evidence that talpid3 embryos can be used to model human ciliopathy mutations, demonstrate that PGCs are a primary cell model that can be used to study the centrosome and identify CCDC127 as a novel centrosome protein that is necessary for ciliogenesis in human RPE1 cells. Additionally, the findings show that protein pathways associated with the regulation of the actin cytoskeleton are downregulated in talpid3. Together the results produced in this thesis provide insight into the centrosome from the perspective of chicken PGCs as well as a better understanding of protein pathways altered in talpid3.

With continuing advances in genome sequencing technology, the chicken genome assembly is now better annotated with improved accuracy to the level of single nucleotide polymorphisms. Additionally, the genomes of other birds such as the duck, turkey and zebra finch have now been sequenced. A great opportunity exists in avian biology to use genome editing technology to introduce small and defined sequence changes to create specific haplotypes in chicken to investigate gene regulatory function, and also perform rapid and seamless transfer of specific alleles between chicken breeds. The methods for performing such precise genome editing are well established for mammalian species but are not readily applicable in birds due to evolutionary differences in reproductive biology. A significant leap forward to address this challenge in avian biology was the development of long-term culture methods for chicken primordial germ cells (PGCs). PGCs present a cell line in which to perform targeted genetic manipulations that will be heritable. Chicken PGCs have been successfully targeted to generate genetically modified chickens. However, genome editing to introduce small and defined sequence changes has not been demonstrated in any avian species. To address this deficit, the application of CRISPR/Cas9 and short oligonucleotide donors in chicken PGCs for performing small and defined sequence changes was investigated in this thesis. Specifically, homology-directed DNA repair (HDR) using oligonucleotide donors along with wild-type CRISPR/Cas9 (SpCas9-WT) or high fidelity CRISPR/Cas9 (SpCas9-HF1) was investigated in cultured chicken PGCs. The results obtained showed that small sequences changes ranging from a single to a few nucleotides could be precisely edited in many loci in chicken PGCs. In comparison to SpCas9-WT, SpCas9-HF1 increased the frequency of biallelic and single allele editing to generate specific homozygous and heterozygous genotypes. This finding demonstrates the utility of high fidelity CRISPR/Cas9 variants for performing sequence editing with high efficiency in PGCs. Since PGCs can be converted into pluripotent stem cells that can potentially differentiate into many cell types from the three germ layers, genome editing of PGCs can, therefore, be used to generate PGC-derived avian cell types with defined genetic alterations to investigate the host-pathogen interactions of infectious avian diseases. To investigate this possibility, the chicken ANP32A gene was investigated as a target for genetic resistance to avian influenza virus in PGC-derived chicken cell lines. Targeted modification of ANP32A was performed to generate clonal lines of genome-edited PGCs. Avian influenza minigenome replication assays were subsequently performed in the ANP32A-mutant PGC-derived cell lines. The results verified that ANP32A function is crucial for the function of both avian virus polymerase and human-adapted virus polymerase in chicken cells. Importantly, an asparagine to isoleucine mutation at position 129 (N129I) in chicken ANP32A failed to support avian influenza polymerase function. This genetic change can be introduced into chickens and validated in virological studies. Importantly, the results of my investigation demonstrate the potential to use genome editing of PGCs as an approach to generate many types of unique cell models for the study of avian biology. Genome editing of PGCs may also be applied to unravel the genes that control the development of the avian germ cell lineage. In the mouse, gene targeting has been extensively applied to generate loss-of-function mouse models to use the reverse genetics approach to identify key genes that regulate the migration of specified PGCs to the genital ridges. Avian PGCs express similar cytokine receptors as their mammalian counterparts. However, the factors guiding the migration of avian PGCs are largely unknown. To address this, CRISPR/Cas9 was used in this thesis to generate clonal lines of chicken PGCs with loss-of-function deletions in the CXCR4 and c-Kit genes which have been implicated in controlling mouse PGC migration. The results showed that CXCR4-deficient PGCs are absent from the gonads whereas c-Kit-deficient PGCs colonise the developing gonads in reduced numbers and are significantly reduced or absent from older stages. This finding shows a conserved role for CXCR4 and c-Kit signalling in chicken PGC development. Importantly, other genes suspected to be involved in controlling the development of avian germ cells can be investigated using this approach to increase our understanding of avian reproductive biology. Finally, the methods developed in this thesis for editing of the chicken genome may be applied in other avian species once culture methods for the PGCs from these species are developed.

Primordial germ cells (PGCs) are the precursors of the germ cell lineage that eventually differentiate into mature spermatozoa and oocytes. Although present throughout the animal kingdom, the specification and migration of PGCs differs widely between species. In vertebrates, avians are evolutionary divergent from mammals and therefore allow a comparative system in which to study germ cell development in higher organisms. Unlike mouse, PGCs can be isolated from the chicken embryo, expanded and cultured long term in vitro. Analysis of these cells showed that cultured chicken PGCs maintain the characteristics of their in vivo counterparts, including the expression of key germ cell specific markers and cell surface adhesion proteins, and thus, are an ideal system to study germ cell biology. Further characterisation revealed that an avian homologue of the zinc finger transcription factor PRDM14, essential for the specification of the mammalian germ cell lineage, was expressed in chicken PGCs. cPRDM14 was found to be expressed in PGCs in vitro and in vivo from early developmental stages until expression is lost by embryonic day 10 and subsequently re-expressed in the adult testis. The expression of cPRDM14 suggested that this gene may play a conserved role in the avian germ cell lineage. To investigate the function of cPRDM14, a novel single piggyBac transposon vector containing a reverse tetracycline activator protein and a tetracycline response element-regulated promoter was developed. Testing of the integrated transposon revealed that expression was tightly regulated and it was possible to conditionally express one gene product whilst simultaneously reducing the expression of a second gene, both in vitro and in vivo. This vector system was fully functional in PGCs, and was used to create transgenic founder chickens capable of having gene expression manipulated in germ cells at various developmental stages. Transgenic offspring were produced and the transgene was inducible at early developmental stages in the G1 animals. The un-induced transgene proved to be toxic to early embryos so a transgenic line of birds could not be produced. The inducible transposon was used to knockdown cPRDM14 expression in chicken PGCs. Knockdown of this gene led to reduced PGC numbers and increased cell death, both in vitro and in ovo. Expression of the pluripotency factor cNANOG was also significantly reduced which may explain the increased cell death. The knockdown of cPRDM14 also led to an increased susceptibility of PGCs to spontaneously de-differentiate to pluripotent embryonic germ cells (EGCs). cPRDM14 knockdown PGCs exhibited elevated levels of phosphorylated ERK, a target of the FGF signalling pathway. It was possible to prevent de-differentiation of the knockdown PGCs by removing ectopic FGF from the media. Furthermore, a sustained high level of FGF signalling in the media was sufficient to drive the de-differentiation of control PGCs to EGCs, suggesting that increased FGF signalling was key to the de-differentiation process. Extensive epigenetic remodelling of mouse PGCs occurs during embryonic development and PRDM14 was shown to be involved in this process. Chicken PGCs in vitro, contain several key histone modifications (H3K4me3, H3K9me2 and H3K27me3) and are 5-methyl cytosine (5-mC) positive. Immunohistochemical analysis of these markers in PGCs, at various stages during early embryonic development, suggests that these cells do not undergo the extensive epigenetic remodelling found in their mammalian counterparts. In contrast to the mouse germ cell lineage, knockdown of cPRDM14 in cultured PGCs had no noticeable effect on the epigenetic status of chicken PGCs. Together these results demonstrate that cPRDM14 is essential for the survival and maintenance of germ cell identity in chicken PGCs, but may not be critical for maintaining the epigenetic status of these 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.