Conditional Knockout of WT1 in Sertoli Cells In Vivo

描述（由申请人提供）：WT 1是一种转录因子，最初发现是因为其与遗传获得性儿童异常（包括Wilms肿瘤）相关。后来发现WT 1是一种肿瘤抑制基因，如果在人类的两个等位基因上发生突变，就会增加患多种癌症的可能性。此外，WT 1对于男性生殖道的正常发育至关重要，因为WT 1突变的人患有各种男性性腺异常。在小鼠中，WT 1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，然后具有指导雄性性腺形成的后期胚胎作用。有趣的是，WT 1在出生后的睾丸支持细胞中也有高水平表达，但其功能尚不清楚，因为WT 1基因缺失的小鼠在胚胎发育过程中死亡。在这项提案中，WT 1在出生后和成人支持细胞中的功能将使用一种新的工具进行评估。该工具是Pem同源框基因近端启动子（Pem Pp），其在出生后和成人支持细胞中选择性地以高水平表达。这种特异性是有价值的，因为它意味着该启动子可用于选择性地在支持细胞中表达Cre，从而在这些细胞中选择性地敲除WT 1基因，并在其他地方保留WT 1功能，使得小鼠可以存活至生殖年龄。通过检查切除出生后支持细胞中WT 1基因的表型结果，可以确定WT 1在精子发生中的作用。因为不同长度（0.3和0.6kb）的Pem Pp 5 '-侧翼序列在出生后不同年龄提供表达的激活，这将允许在出生后不同时间敲除支持细胞中的WT 1。通过在Pem Pp中包含lac阻遏物（lacR）序列将产生表达的进一步多功能性，允许该启动子在出生后的不同发育时间响应于lacR去阻遏物IPTG而开启。除了通过基因切除敲除WT 1外，其功能也将被体内从Pem Pp表达的显性阴性WT 1分子抑制。虽然该项目仅致力于研究WT 1基因的功能，但为实现这一目标而制造的转基因小鼠（例如，那些表达来自Pem Pp的Cre的细胞）将是选择性敲除出生后支持细胞中其它基因的宝贵资源。

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.