Next generation transgenic technologies for the chick

The chick embryo is a well-established model for the study of vertebrate development, particularly because it is possible to access the embryos in ovo and carry out experimental manipulations that include grafting of embryonic material, application of growth factors, transient transfection with gene constructs by electroporation or infection with retroviral vectors. The chicken is also a model species for investigation of the general physiology of birds, for example photoperiodism and seasonality. The domestic fowl is of course an important agricultural species and there is significant research in the UK into aspects of the genetics and physiology of the chicken relevant to poultry breeding and production. A significant tool that was missing from the repertoire of technologies available to researchers studying the chicken was the ability to make genetic modifications. We established an effective method for production of transgenic birds, by injection of lentiviral vectors into chick embryos in new laid eggs, followed by culture of injected embryos to hatch. We are proposing additional development of transgenic tools for genetic modification of the chick. Our aim is to improve the efficiency of production of transgenic birds and demonstrate that we can use novel tools to investigate gene function directly in birds. These advances will support research in basic biology of vertebrate development through to understanding aspects of disease susceptibility and resistance in poultry.

We have reproduced the method published recently that allows proliferation of chick primordial germ cells (PGCs) in culture for prolonged periods. This method forms the basis of a cell-based method for production of transgenic chickens. Cultured PGCs can be transfected with transgene constructs, injected into early chick embryos in ovo and identified as forming functional gametes in the resulting chickens. The published methods for stable transfection of PGCs suggest that this is very challenging, with very low frequencies of identification of stably genetically-modified PGCs. We have shown that the piggyBac transposon vector system can be used to generate stable transgenic PGCs very efficiently. Here we propose to show that PGCs selected for integration of piggyBac transposons can form functional gametes following injection into chick embryos. We will also use the piggyBac transposon system to introduce an inducible knockdown miRNA transgene, to demonstrate the use of the tet-inducible transgene expression module and knockdown of a gene in vivo.

The aim of this project is to increase the tools available for genetic modification of the chicken. These improvements will be of value to a number of projects at the Roslin Institute and to the many researchers who work in the chick, from basic developmental biology to poultry breeders. Transgenesis is a tool for studying gene function in vivo and so is useful for the study of most aspects of avian biology. The chicken is a model organism that is studied as a model for vertebrate development but also as a model for avian physiology. In addition, much research, especially in innate immunity, immunology and growth, is highly relevant to poultry breeding. We have very good contacts already established with the UK developmental biology community. This community will be informed by direct contact and also by presentations at the British Society for Developmental Biology conferences. We have also presented at Poultry Science Association meetings and at farm animal health science meetings, which are attended by academics and animal breeding and animal health company researchers. The profile of transgenic research in the chicken at Roslin has a high profile, evidenced by frequent requests for collaboration, supply of resources and advice. We will continue to carry out the activities listed above and also increase our presence on The Roslin Institute website by setting up a Transgenic Chicken Facility page. We also contribute to debate on GM. For example Helen sang has been an advisor to European Food Safety Authority Panel on GM animals.