ES cells provide a new and needed resource for biomedical research. The applicants propose to generate enabling technology to help realize the full potential of this system. ES cells are capable of differentiating in vitro into a wide variety of cell types including hematopoietic, endothelial, lymphoid, cardiac, skeletal muscle, and neural cells. To fully exploit the system, it will be essential to obtain pure lineages entirely in vitro starting with ES cells. The applicants will generate a panel of mAbs that are specific to the major lineages of the early embryo and recognize cell surface antigens. Utilizing these mAbs, the applicants will sort cultured ES cells so as to establish purified cultures of ectoderm, mesoderm, or endoderm cells. In a parallel effort, the applicants will purify the mesodermal precursor utilizing ES cell genetics. Gene targeting will be employed to generate an ES line with a neomycin resistance gene inserted in the brachyury gene expressed specifically in early mesoderm lineages. Utilizing this line, the applicants will generate cultures of early mesoderm cells. The enabling tools resulting from this application provide an essential step towards a future resource consisting of a repository of large numbers of genetically engineered ES cell lines.
