Conditional Knockout of WT1 in Sertoli Cells In Vivo

描述（由申请人提供）：WT1是一种转录因子，最初被发现是因为它与遗传获得性儿童异常有关，包括Wilms肿瘤。人们已经发现，WT1是一种肿瘤抑制基因，如果人类在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，WT1对于男性生殖道的正常发育至关重要，因为携带WT1突变的人类患有各种男性性腺异常。在小鼠中，WT1在未分化性腺发生前的早期发育阶段是必需的，然后在胚胎后期指导雄性性腺的形成。有趣的是，出生后，WT1在睾丸的支持细胞中也有高水平表达，但由于WT1缺失的小鼠在胚胎发生期间死亡，因此无法辨别其功能。在本研究中，我们将使用一种新的工具来评估WT1在出生后和成体支持细胞中的功能。这个工具是Pem同源盒基因近端启动子（Pem Pp），它在出生后和成年的支持细胞中选择性地高水平表达。这种特异性是有价值的，因为这意味着该启动子可以在Sertoli细胞中选择性地表达Cre，从而在这些细胞中选择性地敲除WT1基因，并在其他地方保留WT1功能，从而使小鼠能够存活到生殖年龄。通过检查在出生后支持细胞中切除WT1基因的表型后果，可以确定WT1在精子发生中的作用。由于不同长度（0.3和0.6 kb）的Pem Pp 5’侧序列在出生后的不同年龄激活表达，这将允许在出生后的不同时间敲除支持细胞中的WT1。通过在Pem - Pp中包含lac抑制因子（lacR）序列，进一步的多功能性表达将产生，允许该启动子在出生后的不同发育时期响应lacR抑制因子IPTG而被打开。除了通过基因消融敲除WT1外，其功能也会被体内Pem - Pp表达的显性阴性WT1分子所抑制。虽然该项目仅致力于研究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，从Pem Pp中表达Cre的小鼠）将成为选择性敲除出生后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.