Use of primordial germ cell based chicken transgene & knockout techniques to establish animal models for studies in development & infection
Final Report Abstract
The chicken has a long history as one of the most important model organisms in the field of developmental biology and immunology. B-Lymphocytes were first discovered as antibody producing cells in the chicken and named after the bursa of Fabricius. The first attenuated vaccine was developed by Louis Pasteur against fowl cholera caused by Pasteurella multocida. These are only two examples of the major contributions chickens made to today’s knowledge. In addition, chickens supply about one-thirds of the animal protein in human diets throughout the world and it is estimated that by 2020 chicken meet will be the number one source of animal protein. Because of that diseases like Marek’s Diseases, infectious Bursal Disease and Avian Leucosis Virus infections have a huge impact on the poultry industry. A better understanding of the humoral and cell mediated immune response in poultry is needed to improve current vaccination strategies. Beside that zoonotic diseases like avian influenza or Salmonella infections are a threat for humans and poultry. Chickens resistant to avian influenza could result in a huge benefit for human health. While transgene and especially gene targeting technology is available in mice and livestock species like swine, goat and cattle knockout technology was not available in chickens. It was possible to generate transgenic chickens by DNA microinjection or lentiviral gene transfer. More recently it became feasible to culture chicken primordial germ cells (PGC). PGC can be cultured, transfected, clonally selected and be re-introduced into the embryo where they colonize the gonad and give rise to fully transgenic progeny in the next generation. PGC can be grown in vitro for periods over 150 days and remain germline competent providing a nearly limitless resource of cells. The ability to culture PGC that remain germline competent once they are injected back into the embryo is unique to the chicken. In this project for the first time cell-based homologous recombination in a non-mammalian vertebrate was used to successfully generate the first targeted deletion of a gene in chickens. The target of the gene knockout was the JH segment of the immunoglobulin heavy chain. By deletion of the JH segment recombination of the variable region of the heavy chain of the B cell receptor (BCR) should not be possible. B cells without a functional heavy chain should not be able to express a BCR because the immunoglobulin light chain alone cannot get on the cell surface of the B cell. The absolute targeting frequency in PGC was about one correct targeted clone per 107 transfected cells which is comparable to mouse embryonic stem cells. Injection of the knockout PGC led to chickens with a knockout of the JH segment. Southern Blot analyses confirmed the correct targeting of the JH segment and the single integration of the targeting construct. Analyses of the homozygous JH knockout birds showed a block in post-bursal B cell development. Surprisingly B cell precursors without a BCR are able to migrate into the bursa and proliferate in the bursa follicles till hatch. Four weeks after hatch massive lymphocyte depletion and no separation into follicle medulla and cortex was seen. Serum antibodies were absent in JH knockout chickens. The demonstration of making a site-specific change to the chicken genome and generating a targeted gene knockout in an avian species for the first time completes the range of genetic modifications that can be applied to avian models in biomedical research and poultry production.
