Molecular engineering approaches for quantifying hepatic gene activity in embryonic stem cells

Although defined differentiation strategies exist for generating hepatocyte-like cells from mouse embryonic stem (ES) cells, these systems yield a heterogeneous end population, from which it is difficult to assay, purify and quantify cells of interest and understand their gene expression profile. Using non-viral vector plasm ids, we have designed several constructs for exploring the real-time expression levels of alpha feto-protein and albumin. These vectors encode for enhanced green fluorescent protein (EGFP) and are driven by the regulatory elements of the gene of interest Via transient transfection, the DNA reporter plasmids are delivered into the ES cells to monitor gene behavior and the portion of cells that are liver-like. Parallel transfections using constitutive reporters provide a normalization control and yield insight into changes in gene uptake with varied differentiation. Gene behavior is quantified based on flow cytometry and cells of interest can be further purified from the remaining population via a cell sorter. This process enables additional enzymatic assays to be performed on this homogenous group. In addition to providing a vivid tool for understanding specific gene regulatory motifs, the reporters have further application in assaying cell-cell interactions, cell communication and maturation of cellular components.