Inducible RNAi in spermatogonial stem cells

描述（申请人提供）：精原干细胞（SSCs）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。精子发生是一个经典的干细胞依赖性过程，其中SSC功能受外部小生境刺激和内部基因表达控制。目前，调节这些过程中的精原干细胞的机制知之甚少。由于目前没有已知的特异性形态学或分子标记物用于SSC，因此它们不能被分离并且仅通过功能来定义。因此，在体内研究调控SSC生物学功能的内在分子机制极具挑战性。最近，已经开发了一种化学成分确定的培养系统，该系统支持生殖细胞群富集自我更新的SSC持续延长的时间。利用这种培养系统，现在可以确定SSC功能所必需的基因。此外，当与功能性移植结合使用时，可以明确地确定特定基因对体外SSC活性的直接影响。RNA干扰（RNAi）是一种用于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且可诱导的基于载体的RNAi。这种方法有可能大大提高能力，研究内在的分子机制，SSC自我更新和分化。然而，基于载体的RNAi技术尚未在SSC中进行评估。因此，有必要检查诱导型RNA减少精原干细胞中特定基因表达的功效。本研究拟利用小鼠SSC培养系统和功能性移植试验来实现这一目的。提出的三个具体目标是：1）检查基于载体的RNAi沉默SSC中特异性基因表达的功效，2）评估Tet-On系统在SSC中的诱导型RNAi，以及3）检查诱导型RNAi沉默SSC自我更新所必需的基因的功效。在SSC中开发可诱导的RNAi能力将为研究人员研究特定基因在SSC功能中的作用并定义其作用模式提供一个重要工具。利用这种方法获得的信息将为SSC生物学领域增添必要的知识。这些进展将有可能纠正人类男性不育症，并提高经济价值和濒危物种的生育能力。此外，增加知识的内在机制调节SSC功能将增加一般干细胞生物学的理解，并可能适用于其他成人干细胞群体。公共卫生相关性：该项目的拟议实验将导致建立一种新的工具，用于研究调节雄性性腺干细胞功能的机制。使用这种方法获得的知识将大大提高对男性生育力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症，并提高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.