Embryonic stem cells as a platform for analyzing neural gene transcription

There is a need for improved methods to analyze transcriptional control of mammalian stem cell genes. We propose that embryonic stem cells (ESCs) will have broad utility as a model system, because they can be manipulated genetically and then differentiated into many cell types in vitro, avoiding the need to make mice. Results are presented demonstrating the utility of ESCs for analyzing cis-acting sequences using Olig2 as a model gene. Olig2 is a transcription factor that plays a key role in the development of a ventral compartment of the nervous system and the oligodendrocyte lineage. The functional role of an upstream region (USR) of the Olig2 gene was investigated in ESCs engineered at the undifferentiated stage and then differentiated into ventral neural cells with sonic hedgehog and retinoic acid. Deletion of the USR from the native gene via gene targeting eliminates expression in ventral neural cells differentiated in cell culture. The USR is also essential for regulated expression of an Olig2 transgene inserted at a defined foreign chromosomal site. A subregion of the USR has nonspecific promoter activity in transient transfection assays in cells that do not express Olig2. Taken together, the data demonstrate that the USR contains a promoter for the Olig2 gene and suggest that repression contributes to specific expression. The technology used in this study can be applied to a wide range of genes and cell types and will facilitate research on cis-acting DNA elements of mammalian genes.