Conditional Knockout of WT1 in Sertoli Cells In Vivo

体内支持细胞中 WT1 的条件性敲除

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/7185043
• 关键词: 5' Flanking Region; Ablation; Adult; Age; Alleles; Birth; Cells; Childhood; Development; Dominant-Negative Mutation; Embryo; Embryonic Development; Engineered Gene; Event; Genes; Genetic Transcription; Goals; Gonadal structure; Homeobox Genes; Human; Isopropyl Thiogalactoside; Knock-out; Knockout Mice; Knowledge; Lac Repressors; Length; Male Genital Organs; Mus; Mutate; Mutation; Nephroblastoma; Peru; Plasmids; Probability; Proteins; Publishing; Purpose; Reporting; Resources; Role; Site; Specificity; Spermatogenesis; Staging; Testis; Time; Tissues; Transgenes; Transgenic Mice; Transgenic Organisms; Tumor Suppressor Genes; Undifferentiated; WT1 Protein; WT1 gene; X Chromosome; cancer type; day; homologous recombination; in vivo; male; postnatal; promoter; recombinase; reproductive; response; selective expression; sertoli cell; tool; transcription factor

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是最初发现的转录因子，因为它与包括Wilms肿瘤在内的遗传学相关的儿童异常相关。此后，发现WT1是一种肿瘤抑制基因，如果在人类的两个等位基因中突变，则会增加获得多种类型癌症的可能性。此外，WT1对于男性生殖道的适当发展至关重要，因为患有WT1突变的人类患有多种男性性腺异常。在小鼠中，WT1是在发生性别分化之前最早发展的无分化性腺的最早阶段所必需的，然后具有后来的胚胎角色指导雄性性腺的形成。有趣的是，WT1在出生后的睾丸中还以高水平的水平表达，但由于WT1-NULL小鼠在胚胎发生过程中死亡，因此无法辨别其功能。在此提案中，将使用新工具评估WT1在产后和成年Sertoli细胞中的功能。该工具是PEM同源基因近端启动子（PEM PP），该基因在产后和成年Sertoli细胞中有选择性地表达。该特异性是有价值的，因为这意味着该启动子可用于选择性地表达Sertoli细胞中的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，以便小鼠可以生存到生殖时代。通过检查在产后Sertoli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。因为PEM PP 5'-频线序列不同长度（0.3和0.6 kb）提供了出生时不同年龄的表达激活，因此这将允许在出生后不同时间敲除Sertoli细胞中WT1的敲除。表达的进一步多功能性将通过在PEM PP中纳入LAC阻遏物（LACR）序列而产生，从而使该启动子在出生后的不同发育时期因响应型乳杆菌过压IPTG而转旋。除了通过基因消融淘汰WT1外，其功能还将受到从体内PEM PP表达的显性阴性WT1分子的抑制。尽管该项目仅专门研究WT1基因的功能，但为实现这一目标而制定的转基因小鼠（例如，从PEM PP中表达CRE的人）将是选择性地敲除产后Sertoli细胞中其他基因的宝贵资源。