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

基因工程鸡模型：研究淋巴细胞发育和功能的新工具

• 批准号: 268167186
• 负责人: Professor Dr. Benjamin Schusser
• 金额: --
• 依托单位: Professur für Biotechnologie der Reproduktion
• 依托单位国家: 德国
• 项目类别: Independent Junior Research Groups
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/268167186?language=en
• 关键词: Genetically; engineered; chicken; models; novel

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.

鸡是发育生物学、生理学和免疫学研究的理想动物模型。因此，鸡的基因组是第一个被测序的家畜。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，反向遗传技术在鸡体内的功能研究一直没有，直到最近。随着培养生殖系能力的能力，未分化的鸡原始生殖细胞（PGC），终于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区段，导致囊后B细胞发育的阻断和IG的完全缺乏，获得禽类物种中的第一个基因敲除（KO）。B细胞和T细胞形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腔上囊在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仍是未知的。本项目将利用JH-KO鸡对B细胞发育进行深入分析。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。Ig-KO禽类和野生型对照之间的表型差异将与转录组学分析结合使用，以鉴定调节这些发育步骤的因素，并鉴定调节囊卵泡中皮质和髓质分离的分子信号。此外，还将在转基因鸡模型中研究法氏囊后成熟的B细胞系统，以阐明发育途径和淋巴细胞迁移。最后，在转基因和KO鸟类中的过继转移实验结合细胞追踪研究将用于鉴定B记忆细胞，这是鸟类中迄今为止一个谜一样的细胞群体。该项目的第二部分将重点关注鸟类中最丰富的T细胞群体，即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期间和组织稳态中的作用仍然没有得到解决。为了分析g/d T细胞的重要性，需要在鸡中KO该T细胞群体。CRISPR/Cas9基因靶向技术将用于简化鸡PGC中的基因靶向，并删除g/d T细胞。将使用最先进的免疫学工具详细分析g/d T细胞KO鸟的表型。将进行用球虫和沙门氏菌的感染实验以了解g/d T细胞在该g/d T细胞高物种中的作用。总之，该项目将提高我们对原型GALT物种中B细胞生物学的认识，提高非哺乳类脊椎动物中的基因靶向，并阐明g/d T细胞在鸡中的作用。

项目成果

Unraveling the role of B cells in the pathogenesis of an oncogenic avian herpesvirus

• DOI: 10.1073/pnas.1813964115
• 发表时间: 2018-11-06
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Bertzbach, Luca D.;Laparidou, Maria;Kaufer, Benedikt B.
• 通讯作者: Kaufer, Benedikt B.

Blocking of the CXCR4-CXCL12 Interaction Inhibits the Migration of Chicken B Cells Into the Bursa of Fabricius

• DOI: 10.3389/fimmu.2019.03057
• 发表时间: 2020-01-10
• 期刊: FRONTIERS IN IMMUNOLOGY
• 影响因子: 7.3
• 作者: Laparidou, Maria;Schlickenrieder, Antonina;Schusser, Benjamin
• 通讯作者: Schusser, Benjamin