OVARY-SELECTIVE KNOCKOUT OF IGF-I

卵巢选择性敲除 IGF-I

• 批准号: 6191496
• 负责人: ELI Y ADASHI
• 金额: $ 26.91万
• 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/6191496
• 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.

与啮齿动物相关的信息体记录了一个完整的(卵巢) 卵泡内胰岛素样生长因子-I系统，充满 配体(IGF-I)、受体(I型)、IGF结合蛋白(IGFBP4和5)和 IGFBP-4和5-内切酶。根据目前的观点，形状 在很大程度上，通过大量的体外观察，初步和推测 卵泡内生物可利用的IGF-I的强制性自分泌作用是 颗粒细胞中FSH作用的放大。正是这种促卵泡刺激素的作用 支持卵泡内IGF-I假说的IGF-I的性质 构成有腔(FSH依赖)卵泡的强制性决定因素 发展。对这一所谓“放大”假说的严格验证 需要证明体内卵泡内的不可缺少 IGF-I。尽管有报道显示IGF-1基因的女性零突变体 生殖功能障碍，这些初步观察仍然没有定论 鉴于新生儿死亡率很高(90%)，而新生儿死亡人数很少 对老鼠进行了研究。此外，使用系统性(而不是 卵巢特异的)基因打靶技术排除了对 循环相对于卵巢内的相对作用(局部产生) IGF-I在维持卵巢功能中的作用。提供明确的证据 支持或反对“放大”假说，他们提出了一系列 为影响卵巢而设计的体内补充方法(颗粒 细胞)--选择性“敲除”IGF-I。在所有情况下，都将使用 小鼠α-抑制素的启动子，因其在体内已被证实的能力而被选中 以卵巢选择性的方式强烈推动转基因表达。 具体地说，他们建议培育和鉴定经过工程改造的小鼠 过表达大鼠IGFBP-1，从而隔离/耗尽生物可利用性 卵泡内IGF-I携带Cre/loxP驱动的颗粒细胞选择性的小鼠 IGF-1基因缺失；携带三苯氧胺诱导、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的制备及验证 转基因，成功地使IFG-1基因“漂浮”，并产生了 活的和可育的IFG-1--“开花”突变体。由此得出的洞察力 调查可能导致确认或拒绝将IGF-I作为 强制性卵巢内增强器；以及新的广泛适用的开发 旨在实现卵巢选择性缺失的转基因试剂 利息。