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. 1994 May 10;91(10):4303–4307. doi: 10.1073/pnas.91.10.4303

Dicistronic targeting constructs: reporters and modifiers of mammalian gene expression.

P Mountford 1, B Zevnik 1, A Düwel 1, J Nichols 1, M Li 1, C Dani 1, M Robertson 1, I Chambers 1, A Smith 1
PMCID: PMC43773  PMID: 8183905

Abstract

To investigate the activity of candidate regulatory molecules in mammalian 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 constructs to provide cap-independent translation of a selectable marker from fusion transcripts generated following homologous recombination. These promoterless constructs were highly efficient and have been used both to inactivate the stem-cell-specific transcription factor Oct-4 and to introduce a quantitative regulatory modification into the gene for a stem-cell maintenance factor, differentiation-inhibiting activity. In addition, the inclusion of a beta-galactosidase reporter gene in the constructs enabled accurate and sensitive detection of cellular sites of transcription. This has allowed visualization of putative "stem-cell niches" in which sources of elevated expression of differentiation-inhibiting activity were localized to the differentiated cells surrounding colonies of stem cells.

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