A Xenopus Laevis Research Resource for Immunobiology

此次续签申请的目标是保持和进一步发展世界上最全面的 研究资源专门用于非洲爪哇的两栖动物，作为一种多方面的 生物医学和免疫学研究的实验平台，造福全体 科学界。莱维氏X.laevis的兴趣和医学相关性是由于其 免疫系统与人类的免疫系统，在所有发育阶段进行实验的可及性，以及 大量遗传和基因组资源的可用性，MHC定义的无价近交系、克隆和 转基因青蛙(TG)系，以及细胞系、单抗、MHC四聚体、 用于免疫相关基因的标记重组试剂和有效的聚合酶链式反应引物组。这些 不能在市场上买到的动物和试剂需要保存、丰富和提供 向科学界致敬。与以往的建议一样，我们提出了两个主要目标： (1) 保存和推广莱氏X.laevis免疫生物学研究资源 通过坚持下去 生产、管理和向美国和海外的实验室分发动物存栏和试剂。我们会 保持我们近交系和转基因动物的质量、生产力和福利。我们将提供协助、培训和通知 对利用X.laevis进行研究感兴趣的科学家、学生和教育工作者。我们将促进信息的传播， 通过使用我们的网站并通过与其他人互动，获得资源的可访问性、网络和公众意识 非洲爪哇资源国内和国际。 (2) 开发新的方法和产生新的实验动物和试剂 通过 利用莱氏X.laevis推进3个主要领域的研究：(1)转基因青蛙X.laevis品系： 改进基因组注释，产生新的CRISPR/Cas9介导的自交系和自交系 通过敲入长的单链供体模板(LssDNA)，具有可追踪的免疫细胞类型。(2)莱维氏X.laevis 细胞培养系统和试剂：生产转基因和缺陷细胞系，重组标记免疫 试剂和MHC四聚体以及研制体内外VSV-慢病毒转导载体 在非洲爪哇的研究。(3)莱维氏X.laevis传染病与发育实验平台 免疫毒理学：开发具有成本效益和可靠的实验平台来研究感染和 新出现的人类非结核分枝杆菌(NTM)病原体(例如脓肿分支杆菌)的致病性；以及 评估被忽视的具有内分泌干扰活动的水污染物对免疫和人类健康的风险。在……里面 除了维护对非洲爪哇科学界至关重要的研究资源外，该项目还将 开发广泛适用的新方法和技术，以收集对组织和 器官生理学、免疫学和发育生物学。这将有助于科学工作者的努力 在美国国立卫生研究院的帮助下，社区建立了非洲爪哇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.