Dicistronic targeting constructs: reporters and modifiers of mammalian gene expression

Proc Natl Acad Sci U S A. 1994 May 10;91(10):4303-7. doi: 10.1073/pnas.91.10.4303.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

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


  • DNA-Binding Proteins
  • Octamer Transcription Factor-3
  • Pou5f1 protein, mouse
  • Transcription Factors
  • beta-Galactosidase