OVARY SELECTIVE GENES: CRITICAL OVARIAN REGULATORS

卵巢选择性基因：关键的卵巢调节因子

• 批准号: 6181887
• 负责人: ELI Y ADASHI
• 金额: $ 25.59万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-07 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6181887
• 关键词: RNase protection assay; complementary DNA; computer assisted sequence analysis; female; fertility; gene deletion mutation; gene expression; genetic library; granulosa cell; hormone regulation /control mechanism; in situ hybridization; laboratory mouse; molecular biology information system; nucleic acid hybridization; nucleic acid quantitation /detection; nucleic acid sequence; ovary; robotics; sex cycle; subtraction hybridization

Ovary-selective genes constitute a subset of genes characterized by preferential or exclusive ovarian expression. The critical importance of the ovary-selective genes c-mos, ZP-3, GDF-9, and alpha-inhibin to murine ovarian function was established with null mutants which displayed markedly aberrant ovarian phenotypes and consequent infertility. A loss of function mutation in women established the critical role of the FSH receptor in ovarian function. These observations underlie the hypothesis that ovary- selective genes constitute critical molecular determinants of ovarian function. Thus far, the isolation, identification, and characterization of such ovary-selective genes has proceeded on a case-by-case basis. In this application, we propose a systematic approach. In this context, we report preliminary studies on the use of Suppression Subtractive Hybridization to construct an ovary-selective cDNA library. Ongoing screening of 327 clones has yielded 35 putative novel cDNAs lacking homology to any sequence entry deposited in publicly-accessible databases. We also report preliminary studies on the utility of conditional Cre/LoxP technology with an eye to effect ovary-selective gene deletion. Using the igf-1 gene as a testing paradigm, we document its successful "floxing" and selective deletion in the liver following crossing with a Cre transgenic driven by the albumin promoter. Preliminary studies are reported as to the generation of a Cre transgenic driven by the alpha-inhibin promoter in the hope of effecting granulosa cell-selective deletion of any "floxed" gene of interest. To complete the identification and begin the characterization of novel ovary- selective cDNAs, this proposal outlines a series of complementary in vitro and in vivo experiments. Specifically, we propose to: I) complete the identification of ovary-selective cDNAs using high-throughput robotics and microarrayed DNA chip technology, II) isolate and sequence the full-length of novel ovary-selective cDNAs, III) study the hormonal dependence, (cycle) phase-specific expression, and cellular localization of novel ovary-selective genes and IV) assess the functional role of three novel ovary- selective genes by effecting conditional ovarian gene deletion. Insight derived from this investigation may contribute to novel strategies for the promotion of fertility or its control.

卵巢选择性基因构成以优先或排他性卵巢表达为特征的基因子集。 卵巢选择性基因c-mos、ZP-3、GDF-9和α-抑制素对小鼠卵巢功能的至关重要性是通过无效突变体确定的，这些突变体表现出明显异常的卵巢表型和随后的不育。 女性的功能缺失突变确立了 FSH 受体在卵巢功能中的关键作用。 这些观察结果奠定了以下假设的基础：卵巢选择性基因构成卵巢功能的关键分子决定因素。 到目前为止，此类卵巢选择性基因的分离、鉴定和表征都是根据具体情况进行的。 在此应用中，我们提出了一种系统方法。 在此背景下，我们报告了使用抑制消减杂交构建卵巢选择性 cDNA 文库的初步研究。 对 327 个克隆的持续筛选已产生 35 个推定的新型 cDNA，与公开数据库中存储的任何序列条目缺乏同源性。 我们还报告了关于条件性 Cre/LoxP 技术的实用性的初步研究，旨在实现卵巢选择性基因删除。 使用 igf-1 基因作为测试范例，我们记录了其与白蛋白启动子驱动的 Cre 转基因杂交后在肝脏中成功的“floxing”和选择性删除。 据报道，初步研究是由α-抑制素启动子驱动的Cre转基因的产生，希望能够实现颗粒细胞选择性删除任何感兴趣的“floxed”基因。 为了完成新型卵巢选择性 cDNA 的鉴定并开始表征，该提案概述了一系列互补的体外和体内实验。 具体来说，我们建议：I) 使用高通量机器人技术和微阵列 DNA 芯片技术完成卵巢选择性 cDNA 的鉴定，II) 分离和测序新型卵巢选择性 cDNA 的全长，III) 研究新型卵巢选择性基因的激素依赖性、（周期）阶段特异性表达和细胞定位，IV) 评估三种新型卵巢选择性基因的功能作用 通过影响条件性卵巢基因删除来选择卵巢基因。从这项调查中获得的见解可能有助于制定促进或控制生育的新策略。