Use of a selectable reporter for the isolation of mammalian regulatory genes.
A general method is described for isolating the genes encoding differentiation-specific activators of transcription using genetic selection. Employing regulation of the prolactin encoding gene (PRL) as a model, we have shown that the hamster dihydrofolate reductase-encoding gene (dhfr) is an effective dominant selectable reporter in this methodology. The dhfr coding region was ligated to the rat PRL promoter, and the resultant construct was stably transfected into DHFR- Chinese hamster ovary (CHO) cells, where it had little or no activity. Transfection of these cells with plasmid DNA, containing the coding region of a pituitary-specific transcription factor (Pit-1/GHF-1) in a eukaryotic expression vector, resulted in transfectants in which activation of the chimeric construct, pPRLdhfr, had occurred, enabling these cells to be selected on the basis of their DHFR+ phenotype. Our results suggest that this strategy could be used to isolate unknown genes that regulate a variety of differentiated functions.
Morris
A E
AE
Department of Biological Sciences, Columbia University, New York, NY 10027.
Jiang
Y J
YJ
McChesney
R E
RE
Jackson
A E
AE
Bancroft
C
C
Chasin
L A
LA
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002874:Chromosome Mapping; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000032:analysis; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000032:analysis; D015533:Transcriptional Activation; D014162:Transfection
D004268:DNA-Binding Proteins; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D013762:Tetrahydrofolate Dehydrogenase