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GENERATION OF DEVELOPMENTAL MUTANTS IN MICE

小鼠发育突变体的产生

基本信息

批准号：
3326466
负责人：
ELIZABETH J ROBERTSON
金额：
$ 30.29万
依托单位：
HARVARD UNIVERSITY
依托单位国家：
美国
项目类别：
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

项目摘要

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.

这项提议的主要目标是确定起作用的基因。在哺乳动物胚胎发育中的重要作用。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。在第一种情况下，我们将利用大量的转基因小鼠品系。我们计划使用一种快速的原位杂交技术，以最大限度地提高效率检测隐性突变。第二，中国的文化胚胎干细胞(ES细胞)将被用于产生小鼠携带逆转录病毒载体序列的多个拷贝。我们会使用各种分子和遗传筛选方法来识别病毒诱导的显性、隐性和性连锁突变。这些战略应该导致产生一个影响发育的新插入突变的数量进程。此外，我们计划开发新的战略，以产生恢复携带特定基因突变的ES细胞的克隆靶基因。生长因子基因IGF-II将作为模型这些研究的系统。我们将测试几个DNA结构旨在通过同源重组促进基因破坏，并便于选择其中具有这种事件的ES小区发生了。作为补充，我们还计划使用DNA 扩增技术(聚合酶链式反应)用于筛查用于稀有克隆的逆转录病毒感染ES培养群体这些病毒携带一个插入内源性IGF-II基因的前病毒。在……里面这两种方法，特征化的ES细胞克隆将被用于产生嵌合小鼠，从而将突变引入生殖系。希望这些研究将导致发展任何特定基因在生物进化过程中的作用发育中的胚胎可以进行测试。