CORE--TRANSGENIC MOUSE/GENE TARGETING

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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.