Gene targeting of primordial germ cells using TALEN technology to generate the first knockout transgenic chickens

The chicken is used to study how embryos form and is also a major agricultural species. Being able to selectively target genes in chickens will help to understand and improve disease resistance in birds and poultry production. This will also give us new ways to study how chicken embryos form. Sex stem cells can be used to precisely modify the chicken genome. Gene targeting using TALE nucleases (TALENs) promises to be an efficient method to modify sex stem cells to make knockout chickens. We propose to develop this novel technology to efficiently target genes in sex stem cells using TALEN-directed homologous recombination. In this proposal, we will target two genes needed for germ cells to form. Targeted sex stem cells will be injected into host embryos and bred to produce targeted knockout chickens. The knockout chicken produced will be useful to understand how germ cells form and also as a recipient for sex stem cells from frozen avian biobanks for domesticated poultry and potentially for endangered avian species. These advances will support research in basic biology of vertebrate development with applications in understanding disease resistance and fertility in poultry.

The chicken is a model organism for studying vertebrate developmental biology and a major agricultural species. An efficient gene targeting technology will transform the genetic analysis of disease resistance and poultry production traits and open new tools to study developmental biology using the chicken embryo. Cultured chicken primordial germ cells offer a stem cell-based system for precise genetic manipulation of the chicken. Gene targeting using TALE nucleases (TALENs) promises to be an efficient method to modify these cells for the production of knockout chickens. We propose to develop this novel technology to efficiently target genes in primordial germ cells using TALEN-directed homologous recombination. In this proposal, we will target two genes essential for germ cell development. Targeted primordial germ cells will be injected into host embryos and bred to produce targeted genetic knockouts in chicken. The knockout chicken produced will be useful models for germ cell development and also as a recipient for germ cell transfers in germplasm conservation programs for domesticated poultry and potentially for endangered avian species. These advances will support research in basic biology of vertebrate development with applications in understanding aspects of disease susceptibility and resistance in poultry.

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 disease resistance in birds. In addition, much of this 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. Prof Kaiser is highly active in the Avian Immunology Research Group. The profile of transgenic research in the chicken at Roslin has a high profile, evidenced by frequent requests for collaboration, establishment of the NARF, supply of resources and advice. We will continue to carry out the activities listed above and also increase our presence on The Roslin Institute and NARF websites. We also contribute to debate on GM. For example Dr Mike McGrew has participated in TEDEX talks on novel ideas for avian species conservation using GM models.