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

TitleDicistronic targeting constructs: reporters and modifiers of mammalian gene expression.
Publication TypeJournal Article
Year of Publication1994
AuthorsMountford P, Zevnik B, Düwel A, Nichols J, Li M, Dani C, Robertson M, Chambers I, Smith A
JournalProc Natl Acad Sci U S A
Date Published1994 May 10
KeywordsAnimals, beta-Galactosidase, 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

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.

Alternate JournalProc. Natl. Acad. Sci. U.S.A.
PubMed ID8183905
PubMed Central IDPMC43773