The chicken has served as an excellent animal model in developmental biology, physiology and immunology. Consequently, the chicken genome was the first to be sequenced among the domestic animals. Its availability has greatly facilitated the identification of new candidate genes and their functional characterization in vitro. However, revers genetic technologies for functional in vivo studies in chickens have not been available until recently. With the ability to culture germline competent, undifferentiated chicken primordial germ cells (PGC) it finally became possible to generate targeted gene deletions in chickens. The first gene knockout (KO) in an avian species was obtained by targeting the J segment of the immunoglobulin (Ig) heavy chain (JH) leading to a block in post bursal B cell development and a complete lack of Ig. B cells and T cells form the basis of the adaptive immune system. While in humans B cells mature in the bone marrow, B cells in the chicken mature in the bursa of Fabricius. Even though the essential role of the bursa in B cell development was uncovered decades ago the regulatory mechanisms controlling B cell maturation in birds are unknown. This project will make use of JH-KO chickens for in-depth analyses of B cell development. The importance of Ig gene rearrangement and cell surface expression during various developmental steps will be investigated. Phenotypic differences between Ig-KO birds and wild type controls will be utilized, in combination with transcriptomic analyses, to identify factors regulating these developmental steps and to identify molecular signals regulating segregation of cortex and medulla in bursal follicles. Furthermore, post bursal maturation of the B cell system will be studied in transgenic chicken models to shed light on the developmental pathways and lymphocyte migration. Finally, adoptive transfer experiments in transgenic and KO birds in combination with cell tracking studies will be used to identify B memory cells, a thus far enigmatic cell population in birds. The second part of the project will focus on the most abundant T cell population in birds, the gamma/delta (g/d) T cells. Like in swine and ruminants the role of this lymphocyte population during infections and in tissue homeostasis is still unresolved. In order to analyze the importance of g/d T cells a KO of this T cell population in chickens is needed. CRISPR/Cas9 gene targeting technology will be used to simplify gene targeting in chicken PGC and to delete g/d T cells. The phenotype of g/d T cell KO birds will be analyzed in detail using state of the art immunological tools. Infection experiments with Coccidia and Salmonella will be performed to understand the role of g/d T cells in this g/d T cell high species. In summary, this project will improve our knowledge of B cell biology in a prototype GALT species, improve gene targeting in non-mammalian vertebrates and clarify the role of g/d T cells in chickens.