Dicistronic targeting constructs: reporters and modifiers of mammalian gene expression

P Mountford, B Zevnik, A Düwel, J Nichols, M Li, C Dani, M Robertson, I Chambers, A Smith

Research output: Contribution to journalArticlepeer-review

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.
Original languageEnglish
Pages (from-to)4303-7
Number of pages5
JournalProceedings of the National Academy of Sciences
Volume91
Issue number10
DOIs
Publication statusPublished - 10 May 1994

Keywords

  • Animals
  • Cell Line
  • Cloning, Molecular
  • DNA-Binding Proteins
  • Embryo, Mammalian
  • Gene Expression Regulation
  • Genes
  • Mice
  • Octamer Transcription Factor-3
  • Picornaviridae
  • Plasmids
  • Promoter Regions, Genetic
  • Protein Biosynthesis
  • Restriction Mapping
  • Ribosomes
  • Stem Cells
  • Transcription Factors
  • Transcription, Genetic
  • Transfection
  • beta-Galactosidase

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