GENERATION OF DEVELOPMENTAL MUTANTS IN MICE

这项计划的主要目标是确定基因， 在哺乳动物胚胎发育中的重要作用。 我们的方法将 联合收割机结合了许多实验系统和技术。 两 项目将涉及产生随机插入突变， 小鼠 首先，我们将利用 大量的转基因小鼠品系。 我们计划用一个快速的 原位杂交程序，以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）将被用来产生小鼠 携带逆转录病毒载体序列的多个拷贝。 我们将 使用各种分子和遗传筛选方法， 鉴定病毒诱导显性、隐性和性连锁 突变。 这些战略应导致产生一个 影响发育的新插入突变的数量 过程 此外，我们计划开发新的策略， 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 这些研究的系统。 我们将测试几种DNA结构 被设计为通过同源重组促进基因破坏， 并促进选择其中这些事件已经发生的ES细胞， 发生了。 作为一种补充方法，我们还计划使用DNA 扩增技术（聚合酶链反应）筛选 罕见克隆的逆转录病毒感染ES培养物群体 其携带插入内源性IGF-II基因的前病毒。 在 这两种方法，表征的ES细胞克隆将用于 产生嵌合小鼠，从而将突变引入到 生殖细胞。 希望这些研究能帮助我们 任何特定基因的作用， 胚胎发育可以测试。

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.