Conditional Knockout of WT1 in Sertoli Cells In Vivo

体内支持细胞中 WT1 的条件性敲除

基本信息

批准号：
7185043
负责人：
Michelle Ann Barton
金额：
$ 32.21万
依托单位：
UNIVERSITY OF TX MD ANDERSON CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-03-01 至 2010-02-28
项目状态：
已结题

项目摘要

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.

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性儿童异常（包括肾母细胞瘤）有关。后来发现，WT1 是一种肿瘤抑制基因，如果人类的两个等位基因发生突变，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突变的人类会患有多种男性性腺异常。在小鼠中，WT1是性分化发生之前未分化性腺发育的最早阶段所必需的，然后在后期胚胎中发挥指导雄性性腺形成的作用。有趣的是，WT1在出生后也在睾丸支持细胞中高水平表达，但其功能尚无法辨别，因为WT1缺失的小鼠在胚胎发生过程中死亡。在该提案中，将使用新工具评估 WT1 在出生后和成年支持细胞中的功能。该工具是 Pem 同源盒基因近端启动子 (Pem Pp)，它在出生后和成年支持细胞中选择性高水平表达。这种特异性很有价值，因为这意味着该启动子可用于在支持细胞中选择性表达 Cre，从而选择性地敲除这些细胞中的 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育年龄。通过检查在出生后支持细胞中消除 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' 侧翼序列可在出生后不同年龄激活表达，这将允许在出生后不同时间敲除支持细胞中的 WT1。通过在 Pem Pp 中包含 lac 阻遏物 (lacR) 序列，将产生进一步的表达多功能性，从而允许该启动子在出生后的不同发育时间响应 lacR 去阻遏物 IPTG 而打开。除了通过基因消融来敲除WT1之外，其功能也会受到体内Pem Pp表达的显性失活WT1分子的抑制。尽管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 表达 Cre 的小鼠）将成为选择性敲除出生后支持细胞中其他基因的宝贵资源。