CORE--TRANSGENIC MOUSE/GENE TARGETING

转基因小鼠/基因靶向core将通过以下方式产生转基因小鼠 原核注射携带特定调控基因的构建物 在软骨和骨骼中表达的序列，融合到记者手中 吉恩。转基因小鼠的创建者将被交付给个体 计划调查人员进行后续分析。四个项目中的三个 提出用一系列基因构建产生转基因小鼠。在……里面 此外，这个核心将产生携带突变的ES细胞系 项目调查人员研究的软骨和骨骼发育基因。 由项目调查人员产生的基因靶向构建体将是 电击入小鼠ES细胞并在选择性条件下培养 以丰富同源重组事件。由此产生的ES殖民地 将被挑选、展开和拆分以进行临时冻结和DNA 准备工作。DNA将被隔离并使用适当的 酵素。受限制的DNA随后将被交付给项目 调查人员进行后续的Southern分析。一旦确定，核心 将扩大在氮气中冷冻的目标克隆。核心将会 将携带靶向突变的ES克隆注射到囊胚中 产生老鼠嵌合体。由此产生的小鼠嵌合体将被交付 提供给个别项目调查员，以供后续分析。每一个 这四个项目建议培育出含有靶向突变的小鼠。 转基因小鼠/基因靶向核心是一个必不可少的组成部分 本计划项目的一部分。

The Transgenic mouse/gene targeting core will generate transgenic mice by pronuclear injection of gene constructs carrying specific regulatory sequences that are expressed in cartilage and bone, fused to a reporter gene. The transgenic mouse founders will be delivered to the individual Program investigators for subsequent analysis. Three of the four projects propose to generate transgenic mice with a series of gene constructs. In addition, this core will generate ES cell lines carrying mutations in the cartilage and bone development genes studied by the Program investigators. Gene targeting constructs generated by the Program investigators will be electroporated into mouse ES cells and cultured under selective conditions to enrich for homologous recombination events. The resulting ES colonies will be picked, expanded, and split for temporary freeze and DNA preparation. DNAs will be isolated and restricted with the appropriate enzymes. The restricted DNAs will then be delivered to the Project investigators for subsequent Southern analysis. Once identified, the core will expand targeted clones for freeze down in nitrogen. The core will inject ES clones carrying the targeted mutations into blastocysts to generate mouse chimeras. The resulting mouse chimeras will be delivered to the individual program investigators for subsequent analysis. Each of the four projects proposes to generate mice containing targeted mutations. The Transgenic mouse/gene targeting core represents an essential component of this Program Project.