OVARY-SELECTIVE KNOCKOUT OF IGF-I

卵巢选择性敲除 IGF-I

• 批准号: 6637970
• 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/6637970
• 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（IGF）-I系统，充满 配体（IGF-I）、受体（I型）、IGF结合蛋白（IGFBPs 4和5）和 IGFBP-4和5定向内肽酶。根据目前的观点， 在很大程度上，通过大量的体外观察，主要的和可能的 生物可利用的卵泡内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 IGF-I的性质，这是卵泡内IGF-I 构成了一个强制性的决定因素，窦（FSH依赖）卵泡 发展对这个所谓的“放大”假说的严格验证 需要证明体内滤泡内 IGF-I。尽管有报道称IGF-1基因的雌性无效突变体 生殖功能障碍，这些初步观察仍然是不确定的 鉴于新生儿死亡率高（<90%）， 老鼠研究此外，使用系统（而不是 卵巢特异性）基因打靶技术排除了确定 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 不管是支持还是反对“放大”假说，他们提出了一系列 设计用于影响卵巢（颗粒）的互补体内方法 细胞）选择性“敲除”IGF-I。在所有情况下，将使用 小鼠α-腺苷酸结合蛋白的启动子，选择它是因为它在体内被证明能够 以卵巢选择性方式强烈驱动转基因表达。 具体地说，他们建议产生和表征基因工程小鼠， 过表达大鼠IGFBP-1，从而隔离/耗尽生物可利用的 卵泡内IGF-I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的Cre/loxP驱动的 颗粒细胞选择性IGF-1基因缺失。初步数据包括 生成和验证α-β-bin/IGFBP-1和α-β-bin/Cre 转基因，成功的ifg-1基因的“floxing”，以及 可存活和可育的IFG-1-“floxed”突变体。由此得出的见解 研究可能导致确认或拒绝IGF-I作为 强制性卵巢内放大器;和开发新的广泛适用的 设计用于实现卵巢选择性缺失以下基因的转基因试剂： 兴趣