Inducible RNAi in spermatogonial stem cells

描述（由申请人提供）：精原干细胞（ssc）的自我更新和分化为持续的精子发生提供了基础。因此，雄性的持续生育能力和遗传系的保存严重依赖于SSC的生物学功能。精子发生是一个典型的干细胞依赖过程，其中SSC的功能受外部生态位刺激和内部基因表达的控制。目前，对ssc中调节这些过程的机制了解甚少。由于目前还没有已知的ssc特异性形态或分子标记，因此它们不能被分离出来，只能通过功能来定义。因此，在体内研究调节SSC生物学功能的内部分子机制是极具挑战性的。最近，一种化学定义的培养系统已经被开发出来，它支持生殖细胞群在很长一段时间内丰富自我更新的ssc。利用这种培养系统，现在可以确定对SSC功能至关重要的基因。此外，当与功能移植结合使用时，可以明确确定特定基因对体外SSC活性的直接影响。RNA干扰（RNAi）是破译分子通路和评估特定基因生物学意义的有力工具。最近的技术进步允许在真核细胞中产生稳定和可诱导的基于载体的RNAi。这种方法有潜力极大地提高研究控制SSC自我更新和分化的内在分子机制的能力。然而，基于载体的RNAi技术尚未在ssc中进行评估。因此，有必要研究诱导RNA在ssc中降低特定基因表达的功效。本研究将利用小鼠SSC培养系统和功能移植实验来实现这一目标。提出的三个具体目标是：1)研究基于载体的RNAi沉默SSC中特定基因表达的效果；2)评估SSCs中诱导RNAi的Tet-On系统；3)研究诱导RNAi沉默SSC自我更新所需基因的效果。在SSC中诱导RNAi能力的发展将为研究人员检查特定基因在SSC功能中的作用并确定其作用模式提供重要工具。利用这种方法所获得的信息将为SSC生物学领域增加必要的知识。这些进展将有可能纠正人类男性不育，并提高具有经济价值和濒危物种的生育能力。此外，增加对调节SSC功能的内在机制的了解将增加对一般干细胞生物学的理解，并可能适用于其他成体干细胞群体。公共卫生相关性：本项目拟开展的实验将为研究男性性腺干细胞功能调节机制建立一种新的工具。从使用这种方法中获得的知识将大大提高对男性生育和干细胞生物学的理解。这些进展将有可能纠正人类男性不育，并提高具有经济价值和濒危物种的生育能力。

DESCRIPTION (provided by applicant): Self-renewal and differentiation by spermatogonial stem cells (SSCs) provides the foundation for continual spermatogenesis. Thus, sustained fertility in males and preservation of genetic lines is critically dependent on SSC biological function. Spermatogenesis is a classic stem cell-dependent process in which SSC function is controlled by external niche stimuli and internal gene expression. Currently, mechanisms regulating these processes in SSCs are poorly understood. Because there are currently no known specific morphological or molecular markers for SSCs, they cannot be isolated and are defined solely by function. Therefore, it is extremely challenging to study the internal molecular mechanisms regulating SSC biological function in vivo. Recently, a chemically defined culture system has been developed that supports a germ cell population enriched for self-renewing SSCs for extended periods of time. Utilizing this culture system it is now possible to define the genes that are essential for SSC functions. Moreover, when utilized in conjunction with functional transplantation, direct effects of specific genes on SSC activity in vitro can be unequivocally determined. RNA interference (RNAi) is a powerful tool for deciphering molecular pathways and assessing the biological significance of specific genes. Recent technological advances allow for generation of stable and inducible vector based RNAi in eukaryotic cells. This methodology has potential for dramatically enhancing the ability to study intrinsic molecular mechanisms governing SSC self-renewal and differentiation. However, vector-based RNAi technologies have not been evaluated in SSCs. Thus, it is essential to examine the efficacy of inducible RNA to reduce specific gene expression in SSCs. In this proposal the mouse SSC culture system and functional transplantation assay will be utilized to achieve this objective. The three proposed specific aims are: 1) Examine the efficacy of vector based RNAi for silencing specific gene expression in SSCs, 2) Evaluate the Tet-On system for inducible RNAi in SSCs, and 3) Examine the efficacy of inducible RNAi to silence genes essential for SSC self-renewal. Development of inducible RNAi capabilities in SSCs will add an essential tool for researchers to examine the role of specific genes in SSC functions and define their modes of action. The information gained from utilization of this methodology will add essential knowledge to the field of SSC biology. These advances will have potential for correcting male infertility in humans and enhancing fertility in economically valuable and endangered species. Also, increased knowledge of intrinsic mechanisms regulating SSC function will add to the understanding of general stem cell biology and may be applicable to other adult stem cell populations. PUBLIC HEALTH RELEVANCE: Proposed experiments of this project will lead to establishment of a novel tool for studying mechanisms regulating stem cell functions in the male gonad. Knowledge gained from use of this methodology will dramatically enhance the understanding of male fertility and stem cell biology. These advances will have potential for correcting male infertility in humans and enhancing fertility in economically valuable and endangered species.