Dicistronic targeting constructs: reporters and modifiers of mammalian gene expression.
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.
Mountford
P
P
Agricultural and Food Research Council Centre for Genome Research, University of Edinburgh, Scotland.
Zevnik
B
B
Düwel
A
A
Nichols
J
J
Li
M
M
Dani
C
C
Robertson
M
M
Chambers
I
I
Smith
A
A
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism; D004622:Embryo, Mammalian; D005786:Gene Expression Regulation; D005796:Genes; D051379:Mice; D050814:Octamer Transcription Factor-3; D010849:Picornaviridae / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D015183:Restriction Mapping; D012270:Ribosomes / Q000378:metabolism; D013234:Stem Cells; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000378:metabolism
D004268:DNA-Binding Proteins; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D014157:Transcription Factors; D001616:beta-Galactosidase