OVARY SELECTIVE GENES: CRITICAL OVARIAN REGULATORS

卵巢选择基因是以卵巢优先表达或排他性表达为特征的基因子集。卵巢选择基因c-mos、ZP-3、GDF-9和α-抑制素对小鼠卵巢功能的重要作用是在卵巢表型明显异常并导致不孕的零突变体中确定的。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。这些观察结果支持了卵巢选择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，这种卵巢选择基因的分离、鉴定和表征都是在个案的基础上进行的。在这一应用中，我们提出了一种系统的方法。在此背景下，我们报道了利用抑制消减杂交技术构建卵巢选择性cDNA文库的初步研究。正在进行的327个克隆的筛选已经产生了35个假定的新的cDNA，与公共可访问的数据库中存储的任何序列条目都没有同源性。我们还报告了条件Cre/loxP技术在卵巢选择性基因缺失方面的应用的初步研究。以IGF-1基因为测试范型，我们证明了它与白蛋白启动子驱动的Cre转基因杂交后，在肝脏中成功地“漂浮”和选择性缺失。初步研究了由α-抑制素启动子驱动的Cre转基因的产生，以期实现颗粒细胞选择性删除任何感兴趣的“花”基因。为了完成对卵巢有选择性的新的cDNA的鉴定和鉴定，这项建议概述了一系列补充的体外和体内实验。具体地说，我们建议：i)利用高通量机器人和微阵列DNA芯片技术完成卵巢选择基因的鉴定；ii)分离和测序新的卵巢选择基因的全长；iii)研究新的卵巢选择基因的激素依赖性、周期特异性表达和细胞定位；以及iv)通过影响卵巢基因条件性缺失来评估三个新的卵巢选择基因的功能作用。从这项调查中得出的见解可能有助于促进生育率或其控制的新战略。

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.