Targeted Suppression of the Transcription Factor Sox9 in the Testis by Tissue-Spe

描述（由申请人提供）：转录因子Sox 9在胚胎发生期间对雄性性腺发育至关重要。Sox 9表达也持续存在于一些成年组织中，特别是在睾丸中，但其在出生后的作用尚不清楚，因为传统的Sox 9敲除（KO）小鼠在胚胎发育期间死亡。在本申请中，我建议确定Sox 9在出生后和成年睾丸中的作用。我建议通过使用一种新开发的组织特异性RNA干扰（RNAi）方法来完成这项任务，该方法使用我最近发现的一种工具-一种在睾丸支持细胞中选择性表达的高活性启动子，该细胞是睾丸中Sox 9表达的位点。我们在最近的研究中表明，只有0.6 kb的5 '侧翼序列的近端启动子的RhoxS同源框基因（RhoxS Pp）是足以驱动强表达，特别是在出生后和成人支持细胞在体内发育调节的方式。我建议使用RhoxS Pp产生转基因小鼠，表达短发夹（sh）RNA特异性的Sox 9在体内。除了这项研究提供了关于Sox 9在出生后睾丸中作用的第一个信息外，它还将证明我新开发的体内RNAi方法的原理，我曾经以组织特异性方式敲除Wilms肿瘤1基因（WT 1）。这种RNAi方法将是一种技术进步，因为迄今为止还没有实验室报道开发出用于体内稳定的组织特异性RNAi的普遍适用的方法。我的方法源于天然存在的短RNA-微RNA（miRNA）-从Pol II转录本中产生的方法。在我的方法中，靶向Sox 9的shRNA分子将由Drosha从RhoxS Pp驱动的前体转录物加工，Drosha是一种普遍表达的RNase-III酶，可以加工出天然存在的miRNA。由于Sox 9 shRNA转录物将特异性地从RhoxS Pp表达，我预测它将特异性地抑制睾丸内支持细胞中的Sox 9表达。我推测Sox 9在支持细胞中的功能是指导精子发生过程中的特定事件。至少有两条证据支持我的假设：首先，Sox 9在成人支持细胞中以阶段特异性方式高表达，表明它可能在生殖细胞分化中起关键作用。其次，我最近发现，WT 1，这是已知的是在成人支持细胞中高度表达，并在胚胎发生过程中的性腺发育中起着至关重要的作用，如Sox 9，也是维持精子发生的重要。因此，我认为Sox 9在精子发生中起着重要作用。本申请的具体目的是：（1）利用组织特异性RNAi技术，阐明转录因子Sox 9在成人睾丸支持细胞中的功能。阐明Sox 9在出生后睾丸中的功能可能会导致更好地理解性腺发育不全和性逆转的病理生理学，这些病理生理学通常在Sox 9突变的人类患者中发现。此外，Sox 9在生殖中的功能的揭示可能会导致不孕症的治疗。

DESCRIPTION (provided by applicant): The transcription factor Sox9 is critical for the male gonadal development during embryogenesis. Sox9 expression also persists in some adult tissues, particularly in the testes, but its role after birth is not known, as conventional Sox9 knockout (KO) mice die during embryogenesis. In this application, I propose to determine Sox9's role in the postnatal and adult testes. I propose to accomplish this task by using a newly developed tissue specific RNA interference (RNAi) approach with a tool that I recently identified - a highly active promoter selectively expressed in Sertoli cells, the site of Sox9 expression in the testes. We have shown in our recent studies that only 0.6-kb 5'-flanking sequences from the proximal promoter of the RhoxS homeobox gene(RhoxS Pp) is sufficient to drive strong expression specifically in postnatal and adult Sertoli cells in a developmentally regulated manner in vivo. I propose to use RhoxS Pp to generate transgenic mice that express short hairpin (sh) RNAs specific for Sox9 in vivo. In addition to this study providing the first information on the role of Sox9 in the testes after birth, it will be a proof of principle for my newly developed in vivo RNAi approach that I used to knock down Wilms' tumor 1 gene (WT1) in a tissue-specific manner. This RNAi approach will be a technical advance, as to date no laboratory has reported the development of a generally applicable method for stable tissue-specific RNAi in vivo. My approach stems from the means by which naturally occurring short RNAs - micro RNAs (miRNAs) - are generated from Pol II transcripts. In my approach, a shRNA molecule targeting Sox9 will be processed from a RhoxS Pp-driven precursor transcript by Drosha, a ubiquitously expressed RNase-lll enzyme that processes out naturally occurring miRNAs. Because the Sox9 shRNA transcript will be specifically expressed from the RhoxS Pp, I predict it will suppress Sox9 expression specifically in Sertoli cells within the testes. I hypothesize that Sox9 functions in Sertoli cells to direct specific events during spermatogenesis. There are at least two lines of evidence that support my hypothesis : first, Sox9 is highly expressed in the adult Sertoli cells in a stage-specific manner suggesting that it might have a pivotal role in germ cell differentiation. Second, I recently showed that WT1, which is known to be highly expressed in adult Sertoli cells and plays a crucial role in gonadal development during embryogenesis like Sox9, is also important for maintenance of spermatogenesis. Therefore, I propose that Sox9 plays an important role in spermatogenesis. The specific aim of this application is: (1) to elucidate the function of transcription factor Sox9 in adult Sertoli cells by using tissue-specific RNAi. The elucidation of Sox9 function in postnatal testes may lead to better understanding of the pathophysiology of gonadal dysgenesis and sex-reversal commonly found in human patients with mutated Sox9. In addition, revelation of Sox9 function in reproduction may lead to treatments for infertility.