Germ cell preservation of immunodeficient pigs utilizing embryo complementation approach

项目摘要/摘要 严重联合免疫缺陷病(SCID)猪是一种宝贵的生物医学资源。例如，猪缺乏 RAG2和IL2RG可作为干细胞移植、免疫学和传染病的有用模型 因为它们缺乏所有的主要淋巴细胞(B-T-NK-)。然而，这些模型的应用受到了限制。 因为这些猪由于免疫缺陷而不能通过自然繁殖繁殖；没有生殖细胞 (精子和卵母细胞)可以从这些动物身上产生。体细胞核移植(即克隆)或 骨髓移植可以克服这一缺点，但它劳动强度大，效率低。 胚胎互补(或胚泡互补)技术提供了一个展示两个 或在一只动物身上表现出更多的表型。该项目的目标是建立一个能够 可用于生产任何类型的免疫缺陷猪的生殖细胞。我们的假设是胚胎 两种胚胎类型的互补，每种都带有免疫缺陷和生殖细胞耗尽的表型，将 允许胚胎中的动物拥有有效的免疫系统，同时限制生殖细胞的发展 仅来自免疫缺陷的遗传背景。我们将通过两个具体目标来阐述我们的假设。目的：1. 建立携带修饰的RAG2/IL2RG产生精子的嵌合体猪。AIM2.建立嵌合体猪 产生携带修饰的RAG2/IL2RG的卵母细胞。新型生殖细胞耗竭猪模型，可扩展 我们对生殖细胞发育的理解，也将被产生和描述。通过引入有针对性的 通过使用CRISPR/Cas9系统在胚胎发育过程中进行修改，我们将快速生成这些 在R21的时间范围内建立猪模型，并展示其产生生殖细胞的能力。我们广泛的 在猪中使用CRISPR/Cas9系统的经验，特别是在胚胎发育期间，将确保成功 完成拟议的目标。从项目中获得的知识可以扩大使用 通过建立与其他机构共享生殖细胞的策略来帮助了解免疫缺陷猪 利用防止生殖细胞不同发育阶段的大动物模型研究生殖细胞发育机制 发展。因为猪在生理、解剖和免疫学上与人类相似，所以它们可以 作为临床前转化模型，在生物医学领域具有良好的应用前景。这项提议的成功将使其多样化 可应用于广泛的生物医学研究领域的动物模型，如HIV，X连锁 免疫缺陷、细胞移植治疗和自身免疫性疾病。

Project summary/Abstract Severe combined immunodeficient (SCID) pigs are a valuable resource in biomedicine. For instance, pigs lacking RAG2 and IL2RG can be a useful model in stem cell transplantation, immunology, and infectious diseases because they lack all major lymphocytes (B-T-NK-). However, application of the models has been limited because the pigs cannot be propagated through natural breeding due to their immunodeficiency; no germ cells (spermatozoa and oocytes) can be produced from these animals. Somatic cell nuclear transfer (i.e. cloning) or bone marrow transplantation may be applied to overcome the shortcoming, but it is labor intensive and inefficient. The embryo complementation (or blastocyst complementation) technique provides an opportunity to exhibit two or more phenotypes in a single animal. The objective of this project is to establish a foundation system that can be applied to produce germ cells from any type of immunodeficient pigs. Our hypothesis is that embryo complementation of two embryo types, each carrying immunodeficient and germ-cell depleted phenotype, will allow animals from the embryos to have a functional immune system while restricting germ-cell development only from the immunodeficient genetic background. We will address our hypothesis by two specific aims. Aim1. Establish chimeric pigs that produce spermatozoa carrying modified RAG2/IL2RG. Aim2. Establish chimeric pigs that produce oocytes carrying modified RAG2/IL2RG. Novel germ-cell depleted pig models, which can expand our understanding of germ cell development, will also be produced and characterized. By introducing targeted modifications during embryogenesis through the use of CRISPR/Cas9 system, we will rapidly generate these pig models within the timeframe of R21 and demonstrate their ability to produce germ cells. Our extensive experience in using the CRISPR/Cas9 system in pigs, especially during embryogenesis, will ensure successful completion of the proposed aims. The knowledge obtained from the project can expand the use of immunodeficient pigs by establishing a strategy to share their germ cells with other institutes and help understand mechanism of germ-cell development using large animal models preventing different stage of germ-cell development. Because pigs are physiologically, anatomically, and immunologically similar to humans, they can be a suitable model in biomedicine as a preclinical translational model. The success of this proposal will diversify animal models that can be applied in a wide array of biomedical research areas such as HIV, X-linked immunodeficiency, cell transplantation therapy, and autoimmune diseases.