OVARY-SELECTIVE KNOCKOUT OF IGF-I

卵巢选择性敲除 IGF-I

• 批准号: 6388232
• 负责人: ELI Y ADASHI
• 金额: $ 27万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6388232
• 关键词: binding proteins; developmental genetics; follicle stimulating hormone; gene deletion mutation; gene expression; gene targeting; genetic translation; genetically modified animals; graafian follicles; granulosa cell; growth factor receptors; histogenesis; hormone regulation /control mechanism; inhibin; insulinlike growth factor; laboratory mouse; mutant; natural gene amplification; phenotype; reproductive development; tamoxifen; tissue /cell culture; transfection

A body of information relevant to the rodent documents a complete (ovarian) intrafollicular insulin-like growth factor (IGF)-I system, replete with a ligand (IGF-I), a receptor (type I), IGF binding proteins (IGFBPs 4&5) and IGFBP-4 and 5-directed endopeptidases. According to current views, shaped in large measure by numerous in vitro observations, the primary and presumably obligatory autocrine role of bioavailable intrafollicular IGF-I is the amplification of FSH action in granulose cells. It is this FSH-amplifying property of IGF-I which underlies the hypothesis that intrafollicular IGF-I constitutes an obligatory determinant of antral (FSH-dependent) follicular development. Rigorous validation of this so-called "amplification" hypothesis requires demonstration of the in vivo indispensability of intrafollicular IGF-I. Although female null mutants for the igf-1 gene were reported to display reproductive dysfunction, these preliminary observations remain inconclusive given the high (<90%) neonatal mortality rate and the small (<6) number of the mice studied. Moreover, the employment of systemic (as opposed to ovary-specific) gene targeting technology precluded the determination of the relative role of circulating as opposed to intraovarian (locally-generated) IGF-I in the maintenance of ovarian function. To provide unequivocal evidence for or against the "amplification" hypothesis, they propose a series of complementary in vivo approaches designed to effect ovary (granulose cell)-selective "knockout" of IGF-I. In all cases, use will be made of the promoter of mouse alpha-inhibin, chosen for its proven in vivo ability to strongly drive transgene expression in an ovary-selective fashion. Specifically, they propose to generate and characterize mice engineered to overexpress rat IGFBP-1 and thus to sequester/deplete bioavailable intrafollicular IGF-I. Mice bearing Cre/loxP-driven granulose cell-selective igf-1 gene deletion; and mice bearing Tamoxifen-inducible, Cre/loxP-driven granulose cell-selective igf-1 gene deletion. Preliminary data include generation and validation of alpha-inhibin/IGFBP-1 and alpha-inhibin/Cre transgenics, successful "floxing" of the ifg-1 gene, and the generation of viable and fertile ifg-1-"floxed" mutants. Insight derived from this investigation may result in the confirmation or rejection of IGF-I as an obligatory intraovarian amplifier; and development of novel widely-applicable transgenic reagents designed to effect ovary-selective deletion of a gene of interest.

与啮齿动物相关的完整信息（卵巢） 滤泡内胰岛素样生长因子 (IGF)-I 系统，充满 配体 (IGF-I)、受体（I 型）、IGF 结合蛋白 (IGFBPs 4&5) 和 IGFBP-4 和 5 定向内肽酶。按照目前的看法，形 在很大程度上通过大量的体外观察，主要的并且可能是 生物可利用的滤泡内 IGF-I 的强制性自分泌作用是 颗粒细胞中 FSH 作用的放大。正是这种 FSH 放大作用 IGF-I 的特性是滤泡内 IGF-I 假说的基础 构成窦状（FSH 依赖性）卵泡的强制性决定因素 发展。严格验证这种所谓的“放大”假设 需要证明滤泡内的体内必不可少的 IGF-I。尽管据报道 igf-1 基因的女性无效突变体显示 生殖功能障碍，这些初步观察结果仍不确定 鉴于新生儿死亡率高（<90%）且新生儿数量少（<6） 老鼠研究了。此外，使用系统性的（而不是 卵巢特异性）基因靶向技术排除了 循环相对于卵巢内（本地产生）的相对作用 IGF-I有维持卵巢功能的作用。提供明确的证据 支持或反对“放大”假说，他们提出了一系列 旨在影响卵巢的补充体内方法（颗粒 细胞）选择性“敲除”IGF-I。在所有情况下，都将使用 小鼠 α-抑制素启动子，因其经过验证的体内能力而被选中 以卵巢选择性方式强烈驱动转基因表达。 具体来说，他们建议生成并表征经过改造的小鼠 过度表达大鼠 IGFBP-1，从而隔离/耗尽生物利用度 滤泡内 IGF-I。小鼠携带 Cre/loxP 驱动的颗粒细胞选择性 igf-1基因缺失；以及携带他莫昔芬诱导、Cre/loxP 驱动的小鼠 颗粒细胞选择性 igf-1 基因缺失。初步数据包括 α-抑制素/IGFBP-1 和α-抑制素/Cre 的生成和验证 转基因，ifg-1基因的成功“floxing”，以及产生 存活且可育的 ifg-1-“floxed”突变体。由此得出的见解 调查可能会导致确认或拒绝 IGF-I 作为 强制性卵巢内放大器；并开发出适用范围广泛的新型 设计用于实现卵巢选择性删除基因的转基因试剂 兴趣。