Targeted Suppression of the Transcription Factor Sox9

转录因子 Sox9 的靶向抑制

• 批准号: 7415283
• 负责人: Manjeet Kumar Rao
• 金额: $ 7.3万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7415283
• 关键词: RNA interference; Sertoli cells; cell differentiation; gene induction /repression; genetic promoter element; genetically modified animals; homeobox genes; laboratory mouse; microRNAs; northern blottings; polymerase chain reaction; protein structure function; ribonuclease III; spermatogenesis; technology /technique development; terminal nick end labeling; testis; transcription factor; western blottings

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.

描述（由申请人提供）：转录因子 Sox9 对于胚胎发生过程中的男性性腺发育至关重要。 Sox9 表达也在一些成年组织中持续存在，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 Sox9 敲除 (KO) 小鼠在胚胎发生过程中死亡。在此应用中，我建议确定 Sox9 在出生后和成人睾丸中的作用。我建议通过使用新开发的组织特异性 RNA 干扰 (RNAi) 方法和我最近发现的工具来完成这项任务，该工具是在支持细胞（睾丸中 Sox9 表达的位点）中选择性表达的高活性启动子。我们在最近的研究中表明，RhoxS 同源盒基因 (RhoxS Pp) 近端启动子的 0.6-kb 5' 侧翼序列足以在体内以发育调控的方式在出生后和成年支持细胞中特异性驱动强表达。我建议使用 RhoxS Pp 来生成体内表达 Sox9 特异性短发夹 (sh) RNA 的转基因小鼠。这项研究除了提供了关于出生后睾丸中 Sox9 作用的第一个信息外，它还将为我新开发的体内 RNAi 方法提供原理证明，我用该方法敲除 Wilms 肿瘤 1 基因 (WT1)组织特异性方式。这种 RNAi 方法将是一项技术进步，因为迄今为止还没有实验室报告开发出一种普遍适用的体内稳定组织特异性 RNAi 方法。我的方法源于从 Pol II 转录本生成天然存在的短 RNA（微小 RNA (miRNA)）的方法。在我的方法中，靶向 Sox9 的 shRNA 分子将由 Drosha 从 RhoxS Pp 驱动的前体转录本加工而来，Drosha 是一种普遍表达的 RNase-III 酶，可处理天然存在的 miRNA。由于 Sox9 shRNA 转录物将由 RhoxS Pp 特异性表达，因此我预测它将特异性抑制睾丸内支持细胞中的 Sox9 表达。我假设 Sox9 在支持细胞中发挥作用，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，Sox9 在成年支持细胞中以特定阶段的方式高度表达，表明它可能在生殖细胞分化中发挥关键作用。其次，我最近证明，WT1 在成年支持细胞中高表达，并且与 Sox9 一样在胚胎发生过程中的性腺发育中发挥着至关重要的作用，它对于维持精子发生也很重要。因此，我认为Sox9在精子发生中发挥重要作用。本申请的具体目的是：（1）利用组织特异性RNAi阐明转录因子Sox9在成体支持细胞中的功能。阐明出生后睾丸中的 Sox9 功能可能有助于更好地了解 Sox9 突变的人类患者中常见的性腺发育不全和性逆转的病理生理学。此外，Sox9在生殖中的功能的揭示可能会导致不孕症的治疗。