GENERATION OF DEVELOPMENTAL MUTANTS IN MICE

小鼠发育突变体的产生

• 批准号: 3326467
• 负责人: ELIZABETH J ROBERTSON
• 金额: $ 3.39万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-01 至 1993-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3326467
• 关键词: Retroviridae; autosomal recessive trait; clone cells; developmental genetics; embryonic stem cell; fusion gene; genetic crossing over; genetic manipulation; genetic models; genetically modified animals; in situ hybridization; insulinlike factor; laboratory mouse; lethal genes; mammalian embryology; mutant; provirus; site directed mutagenesis; transposon /insertion element

The major goal of this proposal is to identify genes which play important roles in mammalian embryogenesis. Our approach will combine a number of experimental systems and techniques. Two projects will involve generating random insertional mutations in mice. In the first, we will take advantage of the availability of a large pool of transgenic mouse strains. We plan to use a rapid in situ hybridization procedure to maximize the efficiency of detecting recessive mutations. In the second, cultures of embryonic stem cells (ES cells) will be used to generate mice carrying multiple copies of a retroviral vector sequence. We will use a variety of molecular and genetic screening methods to identify virally-induced dominant, recessive and sex linked mutations. These strategies should result in the generation of a number of new insertional mutations affecting the developmental process. Additionally, we plan to develop novel strategies for generating and recovering clones of ES cells carrying mutations in specific target genes. The growth factor gene IGF-II will serve as a model system for these studies. We will test several DNA constructs designed to promote gene disruption by homologous recombination, and to facilitate selection of ES cells in which such events have occurred. As a complementary approach we also plan to use a DNA amplification technique (polymerase chain reaction) to screen populations of retrovirally infected ES cultures for rare clones which carry a provirus inserted in the endogenous IGF-II gene. In both approaches, characterized ES cell clones will be used to generate chimeric mice, thus introducing the mutations into the germ line. Hopefully these studies will lead to the development of techniques whereby the role of any specific gene in the developing embryo can be tested.

该提案的主要目标是确定发挥作用的基因 在哺乳动物胚胎发生中发挥重要作用。 我们的方法将 结合了许多实验系统和技术。 二 项目将涉及生成随机插入突变 老鼠。 首先，我们将利用 大量转基因小鼠品系。 我们计划使用快速 原位杂交程序可最大限度地提高效率 检测隐性突变。 第二，文化 胚胎干细胞（ES细胞）将用于培育小鼠 携带多个拷贝的逆转录病毒载体序列。 我们将 使用多种分子和基因筛选方法 识别病毒诱导的显性、隐性和性相关 突变。 这些策略应该导致产生 影响发育的新插入突变的数量 过程。 此外，我们计划制定新的策略来产生 并回收携带特定突变的 ES 细胞克隆 目标基因。 生长因子基因IGF-II将作为模型 系统来进行这些研究。 我们将测试几种 DNA 构建体 旨在通过同源重组促进基因破坏， 并促进选择发生此类事件的 ES 细胞 发生。 作为补充方法，我们还计划使用 DNA 扩增技术（聚合酶链式反应）筛选 逆转录病毒感染的 ES 培养物群体用于稀有克隆 其携带插入内源IGF-II基因的原病毒。 在 这两种方法，特征化的 ES 细胞克隆都将用于 产生嵌合小鼠，从而将突变引入 种系。 希望这些研究能够促进发展 的技术，其中任何特定基因的作用 可以测试发育中的胚胎。