Ew Group

Stapleford, United Kingdom
Stapleford, United Kingdom
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Karpala A.J.,CSIRO | Stewart C.,CSIRO | McKay J.,EW Group | Lowenthal J.W.,CSIRO | Bean A.G.D.,CSIRO
Journal of Immunology | Year: 2011

In mammals, Mda5 and RIG-I are members of the evolutionary conserved RIG-like helicase family that play critical roles in the outcome of RNAvirus infections. Resolving influenza infection in mammals has been shown to require RIG-I; however, the apparent absence of a RIG-I homolog in chickens raises intriguing questions regarding how this species deals with influenza virus infection. Although chickens are able to resolve certain strains of influenza, they are highly susceptible to others, such as highly pathogenic avian influenza H5N1. Understanding RIG-like helicases in the chicken is of critical importance, especially for developing new therapeutics that may use these systems. With this in mind, we investigated the RIG-like helicase Mda5 in the chicken. We have identified a chicken Mda5 homolog (ChMda5) and assessed its functional activities that relate to antiviral responses. Like mammalian Mda5, ChMda5 expression is upregulated in response to dsRNA stimulation and following IFN activation of cells. Furthermore, RNA interference-mediated knockdown of ChMda5 showed that ChMda5 plays an important role in the IFN response of chicken cells to dsRNA. Intriguingly, although ChMda5 levels are highly upregulated during influenza infection, knockdown of ChMda5 expression does not appear to impact influenza proliferation. Collectively, although Mda5 is functionally active in the chicken, the absence of an apparent RIG-I - like function may contribute to the chicken's susceptibility to highly pathogenic influenza. Copyright © 2011 by The American Association of Immunologists, Inc.

Tyack S.G.,CSIRO | Tyack S.G.,EW Group | Jenkins K.A.,CSIRO | O'Neil T.E.,CSIRO | And 10 more authors.
Transgenic Research | Year: 2013

Traditional methods of avian transgenesis involve complex manipulations involving either retroviral infection of blastoderms or the ex vivo manipulation of primordial germ cells (PGCs) followed by injection of the cells back into a recipient embryo. Unlike in mammalian systems, avian embryonic PGCs undergo a migration through the vasculature on their path to the gonad where they become the sperm or ova producing cells. In a development which simplifies the procedure of creating transgenic chickens we have shown that PGCs are directly transfectable in vivo using commonly available transfection reagents. We used Lipofectamine 2000 complexed with Tol2 transposon and transposase plasmids to stably transform PGCs in vivo generating transgenic offspring that express a reporter gene carried in the transposon. The process has been shown to be highly effective and as robust as the other methods used to create germ-line transgenic chickens while substantially reducing time, infrastructure and reagents required. The method described here defines a simple direct approach for transgenic chicken production, allowing researchers without extensive PGC culturing facilities or skills with retroviruses to produce transgenic chickens for wide-ranging applications in research, biotechnology and agriculture. © 2013 Springer Science+Business Media Dordrecht.

Bakst M.R.,U.S. Department of Agriculture | Wade A.J.,Ew Group
Avian Biology Research | Year: 2014

The normal developmental sequence of the turkey embryo from the initial cleavage divisions through hypoblast formation has been described previously in 11 separate stages based on the progressive morphological differentiation of the embryo (Gupta and Bakst, Turkey embryo staging from cleavage through hypoblast formation. J. Morphol., 217, 313-325, 1993). However, in recent preliminary studies, our attempts to apply this stage table to describe the stages of embryo development were not successful. Therefore, we re-evaluated the development sequence of the turkey using eggs obtained from modern day commercial lines. Embryos from unincubated eggs and eggs incubated at different time intervals up to 25 hour were examined. In contrast to the observations by Gupta and Bakst, embryos from unincubated eggs lack an area pellucida (AP) but are characterised by dense clusters of cells that do not begin to dissipate and begin forming the AP until after 3-4 hours of incubation. Koller's sickle may or may not be present prior to and during hypoblast formation. Based on these new observations, a revised stage table including the above observations is presented to reflect more accurately the development of the modern commercial turkey embryo.

Rutherford K.,Dalhousie University | Meehan C.J.,Dalhousie University | Meehan C.J.,Institute of Tropical Medicine | Langille M.G.I.,Dalhousie University | And 6 more authors.
Poultry Science | Year: 2016

Transposable elements (TEs), such as endogenous retroviruses (ERVs), are common in the genomes of vertebrates. ERVs result from retroviral infections of germ-line cells, and once integrated into host DNA they become part of the host's heritable genetic material. ERVs have been ascribed positive effects on host physiology such as the generation of novel, adaptive genetic variation and resistance to infection, as well as negative effects as agents of tumorigenesis and disease. The avian leukosis virus subgroup E family (ALVE) of endogenous viruses of chickens has been used as a model system for studying the effects of ERVs on host physiology, and approximately 30 distinct ALVE proviruses have been described in the Gallus gallus genome. In this report we describe the development of a software tool, which we call Vermillion, and the use of this tool in combination with targeted next-generation sequencing (NGS) to increase the number of known proviruses belonging to the ALVE family of ERVs in the chicken genome by 4-fold, including expanding the number of known ALVE elements on chromosome 1 (Gga1) from the current 9 to a total of 40. Although we focused on the discovery of ALVE elements in chickens, with appropriate selection of target sequences Vermillion can be used to develop profiles of other families of ERVs and TEs in chickens as well as in species other than the chicken. © 2016 Poultry Science Association Inc.

Wade A.J.,EW Group | French N.A.,Aviagen | Ireland G.W.,University of Manchester
Poultry Science | Year: 2014

Diseases such as avian influenza can destroy turkey flocks, potentially resulting in the loss of valuable or rare genetic material. Consequently, there is an urgent need to develop a means to archive such germplasm. Germline chimeras produced by intravascular transfer of primordial germ cells (PGC) have been reported in other avian species but not turkeys. This study examined the feasibility of both establishing an archive of frozen PGC, and producing germline chimeras by injecting the thawed PGC into host embryos. To meet these aims, the following experiments were performed: (1) PGC identification within turkey embryos; (2) development of an efficient method for isolation of turkey PGC; (3) demonstration that PGC can be cryopreserved, recovered, and retain viability; (4) reinjection into embryos and detection of injected PGC. Primordial germ cells were identified using periodic acid- Schiff reagent and the immunological marker OLP-1. Bloodstream PGC were isolated using Ficoll density gradient centrifugation with PGC recovery peaking at stages 13, 14, and 15 with 32 ± 4.9, 33 ± 6.4, and 26 ± 5.4 PGC recovered, respectively. Primordial germ cells were frozen using Dulbecco's modified Eagle medium, 20% fetal calf serum, and 10% dimethylsulfoxide and demonstrated 90 ± 1.7% viability after 3 mo frozen in liquid nitrogen. Freshly isolated and frozen thawed DiI- and Q-Tracker-labeled PGC repopulated stage 30 gonads after vascular transfer into ex ovo cultured embryos. The DiI-labeled cells repopulated gonads less frequently, with 36 ± 13.2% of gonads containing the DiI-labeled PGC, and 7 ± 3.8% of reinjected PGC reaching the gonads of positive embryos. The Q-tracker- labeled cells were detected more frequently in embryos, with 67 ± 21.1% having positive signals, and 44 ± 4.9% of reinjected Q-tracker-labeled PGC colonized the gonads of positive embryos. This study demonstrated the feasibility of using turkey PGC to archive turkey germplasm from different strains because frozen PGC reintroduced into host embryos can colonize the host gonads, suggesting the possibility of producing turkey germline chimeras. © 2014 Poultry Science Association Inc.

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