OVARY SELECTIVE GENES: CRITICAL OVARIAN REGULATORS

卵巢选择性基因是一组以卵巢特异性表达为特征的基因。 卵巢选择性基因c-mos、ZP-3、GDF-9和α-glycobin对小鼠卵巢功能的至关重要性是通过无效突变体建立的，这些基因显示出明显异常的卵巢表型和随之而来的不孕症。 女性功能缺失突变确立了FSH受体在卵巢功能中的关键作用。 这些观察结果支持了卵巢选择性基因构成卵巢功能的关键分子决定因素的假设。 到目前为止，这种卵巢选择性基因的分离、鉴定和表征是在个案基础上进行的。 在本申请中，我们提出了一种系统的方法。 在此背景下，我们报告了利用抑制性消减杂交构建卵巢选择性cDNA文库的初步研究。 正在进行的筛选327克隆产生了35个推定的新的cDNA缺乏同源性的任何序列条目存放在公众可访问的数据库。 我们还报告了初步研究的实用性的条件Cre/LoxP技术，着眼于影响卵巢选择性基因删除。 使用IGF-1基因作为测试范例，我们记录了其成功的“floxing”和选择性删除在肝脏中与Cre转基因驱动的白蛋白启动子杂交。 初步研究报告的Cre转基因的α-glycobin启动子驱动下，在颗粒细胞选择性删除任何“floxed”感兴趣的基因的希望的产生。 为了完成新的卵巢选择性cDNA的鉴定并开始表征，该提议概述了一系列补充的体外和体内实验。 具体而言，我们建议：I）使用高通量机器人和微阵列DNA芯片技术完成卵巢选择性cDNA的鉴定，II）分离和测序新的卵巢选择性cDNA的全长，III）研究新的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，以及IV）通过实现条件性卵巢基因缺失来评估三种新的卵巢选择性基因的功能作用。从这项调查中获得的见解可能有助于促进生育或控制生育的新策略。

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.