A Xenopus Laevis Research Resource for Immunobiology

此次更新申请的目标是保持并进一步发展世界上最全面的 研究资源，专门利用两栖动物非洲爪蟾作为多方面的 生物医学和免疫学研究的实验平台，并为整个利益 科学界。X的兴趣和医学相关性。由于它们的显著相似性， 免疫系统与人类的免疫系统相似，在所有发育阶段都可以进行实验， 大量遗传和基因组资源的可用性，宝贵的MHC定义的近交系，克隆和 青蛙的转基因（Tg）系，以及诸如细胞系，单克隆抗体，MHC四聚体， 标记的重组试剂和经验证的免疫相关基因的PCR引物组。这些 需要保存、富集和提供不能商业获得的动物和试剂 向科学界展示。与以前的建议一样，提出了两个主要目标： （一） 保护和促进X。免疫生物学研究资源 通过保持 生产，管理和分发动物股票和试剂在美国和国外的实验室。我们将 保持我们的近交系和Tg动物的质量、生产力和福利。我们将协助、培训和告知 对使用X感兴趣的科学家、学生和教育工作者。用于研究。我们会提供信息， 可访问性，网络和公众意识的资源使用我们的网站，并通过与其他 非洲爪蟾资源国内和国际。 （二） 开发新的方法学和产生新的实验动物和试剂 通过 利用X.在3个主要领域进行研究：（1）转基因X。青蛙的laevis线：到 改进基因组注释，产生新CRISPR/Cas9介导的近交Tg敲除（KO）品系和品系 通过敲入长单链供体模板（lssDNA）来追踪免疫细胞类型。(2)X.爪蟾 细胞培养系统和试剂：生产转染和缺陷细胞系，重组标记免疫 试剂和MHC四聚体以及开发用于体外和体内的VSV-慢病毒转导载体 在Xenopus的研究(3)X.传染病和发育实验平台 免疫毒理学：开发具有成本效益和可靠的实验平台，以研究感染和 新出现的人类非结核分枝杆菌（NTM）病原体的致病性（例如，M.（a）（i） 评估被忽视的具有内分泌干扰活性的水污染物对免疫力和人类健康的风险。在 除了维护对非洲爪蟾科学界至关重要的研究资源外，该项目还将 开发广泛适用于收集组织创新见解的新方法和技术， 器官生理学、免疫学和发育生物学。这将有助于科学家的努力。 在美国国立卫生研究院的协助下，社区建立了非洲爪蟾作为生物医学研究的相关模型。

The goal of this renewal application is to maintain and further develop the world's most comprehensive research resource specializing in the use of the amphibian Xenopus laevis as a multi-faceted experimental platform for biomedical and immunological research and for the benefit of the whole scientific community. Interests and medical relevance of X. laevis are due to the remarkable similarity of its immune system with that of human, the accessibility to experimentation at all developmental stages, as well as the availability of large genetic and genomic resources, invaluable MHC-defined inbred strains, clones and transgenic (Tg) lines of frogs, as well as tools such as cell lines, monoclonal antibodies, MHC tetramers, tagged recombinant reagents and batteries of validated PCR primers for immune-relevant genes. These animals and reagents that are not commercially available need to be preserved, enriched, and made available to the scientific community. As in previous proposals, two major main aims are proposed: (1) Preserving and promoting the X. laevis research resource for immunobiology by keeping on producing, managing and distributing animal stocks and reagents to laboratories in the US and abroad. We will maintain the quality, productivity and welfare of our inbred and Tg animals. We will assist, train and inform scientists, students and educators interested in using X. laevis for research. We will foster the information, accessibility, networking and public awareness of the resource using our web site, and by interacting with other Xenopus resources nationally and internationally. (2) Developing new methodologies and generating new experimental animals and reagents by leveraging X. laevis to advance research in 3 main areas: (1) Genetically modified X. laevis lines of frogs: to improve genome annotation, generate new CRISPR/Cas9-mediated inbred Tg knockout (KO) lines and lines with traceable immune cell types by knocking-in long single stranded donor templates (lssDNA). (2) X. laevis cell culture systems and reagents: to produce transfected and deficient cell lines, recombinant tagged immune reagents and MHC tetramers as well as to develop VSV-lentiviral transducing vectors for in vitro and in vivo studies in Xenopus. (3) X. laevis experimental platforms for infectious disease and developmental immunotoxicology: to develop cost-effective and reliable experimental platforms to study infection and pathogenicity of emerging human non-tuberculosis mycobacterium (NTM) pathogens (e.g., M. abscessus); and assess overlooked risk for immunity and human health of water pollutants with endocrine disruption activity. In addition to maintaining a research resource that is crucial for the Xenopus scientific community, this project will develop new approaches and technologies broadly applicable for gathering innovative insights into tissue and organ physiology, immunology and developmental biology. This will contribute to the efforts of the scientific community assisted by the NIH to establish Xenopus as a relevant model for biomedical research.