CORE--TRANSGENIC MOUSE/GENE TARGETING

转基因小鼠/基因靶向核心将通过将基因构建体显微注射到受精小鼠卵中来产生转基因小鼠，以研究骨骼发育。转基因创始人将被交付给个别项目研究人员进行后续分析。所有这四个项目都提出用一系列基因构建体产生转基因小鼠。此外，该核心将产生携带软骨和骨发育基因突变的ES细胞系，由项目研究人员进行研究。将由项目研究者产生的基因靶向构建体电穿孔至小鼠ES细胞中，并在选择性条件下培养以富集同源重组体。将挑选、扩增并分离所得ES细胞集落，用于临时冷冻和DNA制备。将分离DNA并使用适当的酶限制。然后，限制性DNA将被交付给项目调查人员进行随后的Southern分析。一旦确定，核心将扩大目标克隆在氮气中冷冻下来。Core将靶向ES细胞克隆注入囊胚中以产生小鼠嵌合体。将所得小鼠嵌合体交付给个体项目研究者进行后续分析。所有这些项目都建议产生具有靶向突变的小鼠。转基因小鼠/基因靶向核心是该计划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.