A genetic reporter/selection system for optimization of lineage specific embryonic stem cell differentiation

Enhancing the ability of conventional tissue culture techniques to produce specific differentiated cell types, requires an approach that is based upon manipulation of extrinsic and intrinsic signalling pathways combined with selection techniques. As a first step we have developed a series of genetic reporter / selection constructs, which will permit quantitative analysis of lineage-specific differentiation, critical for optimization of tissue engineering protocols. The reporters impart differential fluorescence and antibiotic resistance to ESC-derived cells; such that cell lines can be created that allow the marking and selection of, and discrimination between, cells that are expressing the gene of interest and the progeny of those cells. Our first target is the T-box transcription factor Brachyury (T), which is transiently expressed by nascent early mesoderm progenitors. Our T reporter cell lines will allow us to visualize and manipulate mesoderm development, enable the isolation and characterization of primitive mesoderm progenitors or mesoderm derived cell lines.