Transplantation of Testis Stem Cells in Large Animals

描述(申请人提供)：拟议研究的长期目标是应用生殖系干细胞移植作为一种新的方法来产生人类和动物疾病的生殖系转基因大动物模型。通过雄性生殖系引入遗传修饰将缩短产生转基因动物模型所需的时间，并提供一种与目前通过体细胞核移植诱导非啮齿动物转基因的方法相比可能更有效的策略。睾丸干细胞在成年男性的干细胞中是独一无二的，因为它是唯一可以分裂并将基因贡献给后代的细胞类型，使其成为基因修改的理想目标。由于猪在生物医学研究中的重要性，猪被用作非啮齿动物的模式物种。由于猪的大小和生理学，在猪身上获得的结果预计也适用于其他大型动物物种和人类。这项修订的更新计划的目的是：1)通过生殖细胞移植促进转基因传播；2)提供高效的猪生殖系干细胞体外繁殖系统，并通过操纵雄性生殖系产生转基因猪模型；3)探索用于修饰和移植的替代供体细胞来源；4)探索猪和非人类灵长类动物的分离睾丸细胞异种移植，作为研究和操纵大型动物和人类精子发生的可访问模型。实验将探索慢病毒和非病毒生殖细胞转导系统，量化生殖细胞枯竭对受体猪供体来源精子生成效率的影响，探索生殖细胞培养的新策略，包括使用SV40大T抗原-VP22融合蛋白系统瞬时刺激生殖细胞增殖，以及诱导多能细胞的生殖细胞分化。在多能细胞和生殖系中表达荧光标记的转基因猪模型的生成将为本项目中开发的策略作为克隆的有效替代方案来生成转基因大型动物模型提供“原则证据”。此外，这一猪模型将为未来旨在获得多能猪干细胞和干细胞分化生殖系的研究提供有价值的资源。将分离的细胞移植到小鼠宿主体内后，功能性睾丸组织的从头形态发生将进一步发展，作为生殖细胞移植到同种受体动物睾丸的补充方法。这一新的方法允许在体内培养系统中从分离的细胞重建精子发生，这一过程目前在体外是不可能的。该系统将作为生殖系干细胞潜力的生物测定，并将能够在精子发生重建之前筛选针对特定睾丸细胞类型的基因变化。总体而言，本提案中将进一步开发的同种生殖细胞移植和睾丸细胞移植的新的、互补的策略将使在大型动物模型中进行有效的生殖系修改成为可能。

DESCRIPTION (provided by applicant): The long-term objective of the proposed research is to apply transplantation of germ line stem cells as a new approach to generate germ line transgenic large animal models of human and animal disease. Introduction of genetic modifications through the male germ line will shorten the time necessary to produce transgenic animal models and provide a strategy that is potentially more efficient compared to the current method to induce transgenesis in non-rodent animals by somatic cell nuclear transfer. The testis stem cell is unique among the stem cells in an adult male in that it is the only cell type that divides and can contribute genes to subsequent generations, making it an ideal target for genetic modifications. The pig is used as a nonrodent model species because of its importance for biomedical research. Due to the size and physiology, results obtained in pigs are expected to be applicable to other large animal species and man. The aims of this revised renewal project are 1) to improve transgene transmission through germ cell transplantation, 2) to provide an efficient system for in vitro propagation of porcine germ line stem cells, and to generate a transgenic pig model through manipulation of the male germ line; 3) to explore alternate sources of donor cells for modification and transplantation; and 4) to explore xenografting of isolated testis cells from pigs and nonhuman primates as accessible models for the study and manipulation of spermatogenesis in large animals and man. Experiments will explore lentiviral and non-viral germ cell transduction systems, quantify effects of germ cell depletion on efficiency of donor-derived spermatogenesis in recipient pigs, investigate novel strategies of germ cell culture, including transient stimulation of germ cell proliferation using an SV40 Large T antigen-VP22 fusion protein system, and induction of germ cell differentiation in pluripotent cells. Generation of a transgenic pig model expressing a fluorescent marker in pluripotent cells and in the germ line will provide 'proof of principle' that the strategy developed in this project serves as an efficient alternative to cloning for the generation of transgenic large animal models. In addition, this pig model will provide a valuable resource for future research aimed at derivation of pluripotent porcine stem cells and for germ line differentiation from stem cells. De novo morphogenesis of functional testis tissue after grafting of isolated cells to mouse hosts will be further developed as a complementary approach to germ cell transplantation to the testes of homologous recipient animals. This novel approach allows reconstitution of spermatogenesis from isolated cells in an in vivo culture system, a process that is currently not possible in vitro. This system will serve as a bioassay for germ line stem cell potential and will enable screening of genetic changes targeted to specific testicular cell types prior to reconstitution of spermatogenesis. Overall, the novel, complementary strategies of homologous germ cell transplantation and testis cell grafting to be further developed in this proposal will enable efficient germ line modification in a large animal model.