Germ cell preservation of immunodeficient pigs utilizing embryo complementation approach

项目概要/摘要 重症联合免疫缺陷（SCID）猪是生物医学上的宝贵资源。例如，猪缺乏 RAG 2和IL 2 RG可以是干细胞移植、免疫学和感染性疾病中有用的模型 因为他们缺乏所有主要的淋巴细胞（B-T-NK-）。然而，这些模型的应用受到了限制 因为猪的免疫缺陷不能通过自然繁殖繁殖，没有生殖细胞， （精子和卵母细胞）可以从这些动物产生。体细胞核移植（即克隆）或 骨髓移植可以用来克服这一缺点，但这种方法劳动强度大，效率低。 胚胎互补（或囊胚互补）技术提供了一个机会， 或者在一只动物中有更多的表型。本项目的目标是建立一个基础系统， 可应用于从任何类型的免疫缺陷猪产生生殖细胞。我们的假设是胚胎 两种胚胎类型的互补，每一种都携带免疫缺陷和生殖细胞耗竭表型， 使动物从胚胎开始就具有功能性免疫系统，同时限制生殖细胞的发育 只有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。目标1. 建立产生携带修饰的RAG 2/IL 2 RG的精子的嵌合体猪。目标2。建立嵌合体猪 产生携带修饰的RAG 2/IL 2 RG的卵母细胞。新的生殖细胞耗尽的猪模型，可以扩大 我们对生殖细胞发育的理解，也将产生和特点。通过引入有针对性的 通过使用CRISPR/Cas9系统在胚胎发生过程中进行修饰，我们将快速产生这些 在R21的时间范围内建立猪模型，并证明其产生生殖细胞的能力。我们广泛 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 实现了提出的目标。从项目中获得的知识可以扩大 免疫缺陷猪通过建立与其他机构分享生殖细胞的策略， 利用大动物模型预防生殖细胞发育的不同阶段 发展由于猪在生理、解剖和免疫学上与人类相似， 在生物医学中作为临床前转化模型是合适的模型。这项提案的成功将使 动物模型，可应用于广泛的生物医学研究领域，如艾滋病毒，X连锁 免疫缺陷、细胞移植疗法和自身免疫性疾病。

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.