CORE--TRANSGENIC MOUSE/GENE TARGETING

转基因小鼠/基因靶向Core将通过将构建的基因微注射到受精卵中产生转基因小鼠，以研究骨骼发育。转基因创始人将被交付给个别项目调查员进行后续分析。这四个项目都提出要产生具有一系列基因结构的转基因小鼠。此外，该核心将产生携带软骨和骨发育基因突变的胚胎干细胞系，由项目调查员进行研究。由项目研究者生成的基因靶向构建体将被电穿孔到小鼠胚胎干细胞中，并在选择性条件下培养以富集同源重组。产生的胚胎干细胞菌落将被挑选、扩增和分裂，用于临时冷冻和DNA制备。DNA将被分离出来并用适当的酶进行限制。限制性DNA随后将交付给项目调查员进行后续的南方分析。一旦确定，核心将扩展目标克隆体，在氮气中冷冻。该公司将把目标胚胎干细胞克隆注入囊胚中，以产生小鼠嵌合体。由此产生的小鼠嵌合体将交付给个别项目调查员进行后续分析。所有的项目都建议产生具有靶向突变的小鼠。转基因小鼠/基因靶向核心是本项目的重要组成部分。

The Transgenic Mouse/Gene Targeting Core will generate transgenic mice by microinjection of gene constructs into fertilized mouse eggs to investigate skeletal development. The transgenic founders will be delivered to the individual Program investigators for subsequent analysis. All 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 cartilage and bone development genes studies by the Program Investigators. Gene targeting constructs generated by the Program Investigators will be electroporated into mouse ES cells and culture under selective conditions to enrich for homologous recombinants. The resulting ES cell colonies will be picked, expanded, and split for temporary freeze and DNA preparation. DNA will be isolated and restricted with the appropriate enzymes. The restricted DNA 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 targeted ES cell clones into blastocysts to generate mouse chimeras. The resulting mouse chimeras will be delivered to the individual Program Investigators for subsequent analysis. All of the projects propose to generate mice with targeted mutations. The Transgenic Mouse/Gene Targeting Core represents an essential component of this Program Project.