DICISTRONIC TARGETING CONSTRUCTS - REPORTERS AND MODIFIERS OF MAMMALIAN GENE-EXPRESSION

To investigate the activity of candidate regulatory molecules in mamma lian embryogenesis, we have developed a general strategy for modifying and reporting resident chromosomal gene expression. The picornaviral internal ribosome-entry site was incorporated into gene targeting cons tructs to provide cap independent translation of a selectable marker f rom fusion transcripts generated following homologous recombination. T hese promoterless constructs were highly efficient and have been used both to inactivate the stem-cell-specific transcription factor Oct-4 a nd to introduce a quantitative regulatory modification into the gene f or a stem-cell maintenance factor, differentiation-inhibiting activity . In ad dition, the inclusion of a beta-galactosidase reporter gene in the constructs enabled accurate and sensitive detection of cellular s ites of transcription. This has allowed visualization of putative ''st em-cell niches'' in which sources of elevated expression of differenti ation-inhibiting activity were localized to the differentiated cells s urrounding colonies of stem cells.