Conditional Knockout of WT1 in Sertoli Cells In Vivo

描述(申请人提供)：WT1是一种转录因子，最初被发现是因为它与包括Wilms瘤在内的儿童遗传畸形有关。后来发现，WT1是一种肿瘤抑制基因，如果在人类的两个等位基因上都发生突变，则会增加患多种癌症的可能性。此外，WT1对男性生殖道的正常发育至关重要，因为携带WT1突变的人患有各种男性性腺异常。在小鼠中，WT1在性分化发生之前的未分化性腺发育的最早阶段是必需的，然后在后来的胚胎中起到指导雄性性腺形成的作用。有趣的是，WT1在出生后在睾丸的支持细胞中也有高水平的表达，但它的功能还不可能被识别，因为WT1缺失的小鼠在胚胎发育过程中死亡。在这项建议中，WT1在出生后和成年支持细胞中的功能将使用一种新的工具进行评估。这个工具是Pem Homeobox基因的近端启动子(Pem PP)，它在出生后和成年的Sertoli细胞中选择性地高水平表达。这种特异性很有价值，因为它意味着这个启动子可以用来在Sertoli细胞中选择性地表达Cre，从而选择性地在这些细胞中敲除WT1基因，并在其他地方保留WT1的功能，从而使小鼠能够存活到生殖年龄。通过检测WT1基因在出生后Sertoli细胞中的表型后果，可以确定WT1在精子发生中的作用。由于不同长度的Pem PP 5‘-侧翼序列(0.3和0.6kb)在出生后不同年龄提供表达激活，这将允许WT1在出生后不同时间在支持细胞中被敲除。在Pem PP中加入lac抑制物(LACR)序列将产生进一步的表达多样性，允许该启动子在出生后不同的发育时间被激活，以响应LACR去抑制物IPTG。除了通过基因切割敲除WT1外，在体内Pem PP表达的显性负性WT1分子也会抑制其功能。虽然这个项目只致力于研究WT1基因的功能，但为实现这一目标而制造的转基因小鼠(例如，那些表达Pem PP中Cre的小鼠)将是选择性敲除出生后Sertoli细胞中其他基因的宝贵资源。

DESCRIPTION (provided by applicant): WT1 is a transcription factor originally discovered because of its association with genetically acquired childhood abnormalities, including Wilms' tumor. It has since been discovered that WT1 is a tumor-suppressor gene that if mutated at both alleles in humans increases the probability of acquiring many types of cancer. In addition, WT1 is critical for the proper development of the male genital tract, as humans with WT1 mutations suffer from a variety of male gonadal abnormalities. In mice, WT1 is required for the earliest stage of development of the undifferentiated gonad before sexual differentiation occurs, and then has a later embryonic role directing formation of the male gonad. Intriguingly, WT1 is also expressed at high levels in Sertoli cells in the testis after birth but its function there has not been possible to discern, as WT1-null mice die during embryogenesis. In this proposal, the function of WT1 in postnatal and adult Sertoli cells will be assessed using a new tool. This tool is the Pem homeobox gene proximal promoter (Pem Pp), which is expressed selectively at high levels in postnatal and adult Sertoli cells. This specificity is valuable, as it means that this promoter can be used to selectively express Cre in Sertoli cells and thereby knockout the WT1 gene selectively in these cells and preserve WT1 function elsewhere so that mice can survive to reproductive age. By examining the phenotypic consequences of ablating the WT1 gene in postnatal Sertoli cells, the role of WT1 in spermatogenesis can be determined. Because Pem Pp 5'-flanking sequences of different length (0.3 and 0.6 kb) provide activation of expression at different ages postnatally, this will allow knockout of WT1 in Sertoli cells at different times after birth. Further versatility of expression will be engendered by inclusion of lac repressor (lacR) sequences in the Pem Pp, allowing this promoter to be turned-on at different developmental times after birth in response to the lacR derepressor IPTG. In addition to knocking out WT1 by gene ablation, its function will also be inhibited by dominant-negative WT1 molecules expressed from the Pem Pp in vivo. Although this project is devoted only to studying the function of the WT1 gene, the transgenic mice made to achieve this goal (e.g., those expressing Cre from the Pem Pp) will be a valuable resource for selectively knocking out other genes in postnatal Sertoli cells.