Genetically engineered chicken models: novel tools to investigate lymphocyte development and function

鸡在发育生物学、生理学和免疫学方面是一种优良的动物模型。因此，鸡的基因组是家畜中第一个被测序的。它的可用性极大地促进了新的候选基因的鉴定及其体外功能表征。然而，用于鸡体内功能研究的逆转录基因技术直到最近才出现。随着能够培养种系胜任的，未分化的鸡原始生殖细胞（PGC），最终有可能在鸡中产生靶向基因缺失。通过靶向免疫球蛋白（Ig）重链（JH）的J段，获得了禽类物种的第一个基因敲除（KO），导致法氏囊后B细胞发育受阻，Ig完全缺失。B细胞和T细胞构成适应性免疫系统的基础。人的B细胞在骨髓中成熟，而鸡的B细胞在法氏囊中成熟。尽管几十年前就发现了法氏囊在B细胞发育中的重要作用，但控制鸟类B细胞成熟的调节机制尚不清楚。该项目将利用JH-KO鸡对B细胞发育进行深入分析。我们将研究Ig基因重排和细胞表面表达在不同发育阶段的重要性。将利用Ig-KO鸟类和野生型对照之间的表型差异，结合转录组学分析，确定调节这些发育步骤的因素，并确定调节法氏囊卵泡皮层和髓质分离的分子信号。此外，将在转基因鸡模型中研究法氏囊后B细胞系统的成熟，以阐明其发育途径和淋巴细胞迁移。最后，在转基因和KO鸟类中进行过继转移实验，结合细胞跟踪研究，将用于鉴定B记忆细胞，这是迄今为止鸟类中一个谜一般的细胞群。该项目的第二部分将集中于鸟类中最丰富的T细胞群，γ / δ (g/d) T细胞。与猪和反刍动物一样，这种淋巴细胞群在感染和组织稳态中的作用仍未得到解决。为了分析g/d T细胞的重要性，需要对鸡的g/d T细胞群进行KO。CRISPR/Cas9基因靶向技术将用于简化鸡PGC的基因靶向和g/d T细胞的删除。g/d T细胞KO鸟类的表型将使用最先进的免疫工具进行详细分析。将进行球虫和沙门氏菌感染实验，以了解g/d T细胞在这种g/d T细胞高种中的作用。综上所述，该项目将提高我们对GALT原型物种B细胞生物学的认识，改进非哺乳动物脊椎动物的基因靶向，阐明g/d T细胞在鸡中的作用。

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.