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种系干细胞移植作为一种新方法来产生人类和动物疾病的种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在非啮齿动物中诱导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特的，因为它是唯一可以分裂并可以为后代贡献基因的细胞类型，使其成为遗传修饰的理想靶点。猪被用作非啮齿类动物的模型物种，因为它对生物医学研究的重要性。由于猪的体型和生理特性，在猪中获得的结果有望适用于其他大型动物物种和人类。本修订的更新项目的目的是：1）通过生殖细胞移植改善转基因传递，2）提供一个有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）探索供者细胞的替代来源以用于修饰和移植;以及4）探索来自猪和非人灵长类动物的分离睾丸细胞的异种移植作为研究和操纵大型动物和人的精子发生的可用模型。实验将探索慢病毒和非病毒生殖细胞转导系统，量化生殖细胞耗竭对受体猪中供体来源的精子发生效率的影响，研究生殖细胞培养的新策略，包括使用SV 40大T抗原-VP 22融合蛋白系统瞬时刺激生殖细胞增殖和诱导多能细胞中的生殖细胞分化。在多能细胞和生殖系中表达荧光标记的转基因猪模型的产生将提供“原则证明”，即本项目中开发的策略可作为用于产生转基因大型动物模型的克隆的有效替代方案。此外，这种猪模型将为未来的研究提供了一个宝贵的资源，旨在多能性猪干细胞的衍生和生殖系分化干细胞。将分离的细胞移植到小鼠宿主后，功能性睾丸组织的从头形态发生将进一步发展为生殖细胞移植到同源受体动物睾丸的补充方法。这种新方法允许在体内培养系统中从分离的细胞重建精子发生，这是目前体外不可能的过程。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遗传变化。总的来说，本提案中进一步开发的同源生殖细胞移植和睾丸细胞移植的新的互补策略将使大型动物模型中的生殖系修饰成为可能。

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.