Transplantation of Testis Stem Cells in Large Animals

描述（由申请人提供）：拟议研究的长期目标是应用生殖系干细胞移植作为产生人类和动物疾病的生殖系转基因大型动物模型的新方法。通过雄性生殖系引入基因修饰将缩短生产转基因动物模型所需的时间，并提供一种比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，因为它是唯一一种分裂的细胞类型，可以为后代提供基因，使其成为遗传修饰的理想目标。猪被用作非啮齿动物的模型物种，因为它对生物医学研究很重要。由于猪的体型和生理特征，在猪身上获得的结果有望适用于其他大型动物物种和人类。本修订更新计划的目的是：1)通过生殖细胞移植改善转基因传播，2)为猪生殖系干细胞的体外繁殖提供有效的系统，并通过对雄性生殖系的操作产生转基因猪模型；3)探索用于修饰和移植的供体细胞的替代来源；4)探索从猪和非人类灵长类动物身上分离的睾丸细胞的异种移植，作为大型动物和人类精子发生研究和操作的可行模型。实验将探索慢病毒和非病毒生殖细胞转导系统，量化生殖细胞耗损对受体猪供体来源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SV40大T抗原- vp22融合蛋白系统短暂刺激生殖细胞增殖，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产生表达荧光标记的转基因猪模型将提供“原理证明”，该项目开发的策略可以作为克隆转基因大型动物模型的有效替代方案。此外，该猪模型将为未来多能猪干细胞的衍生和干细胞的生殖系分化研究提供宝贵的资源。将分离细胞移植到小鼠宿主后的功能睾丸组织的新生形态形成将进一步发展，作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中重建精子发生，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。总之，本研究中进一步发展的新的互补策略——同源生殖细胞移植和睾丸细胞移植，将在大型动物模型中实现有效的生殖系修饰。

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.