Isolation of immediate-early differentiation mRNAs by enzymatic amplification of subtracted cDNA from human endothelial cells.
The phenomena of cell growth and differentiation result from unique transcriptional programs. Inducible genes are therefore differentially expressed in response to various biochemical stimuli that regulate cell division and differentiation. To isolate differentially expressed mRNAs of unknown sequence, two techniques, differential and subtractive hybridization, have been used. Subtractive hybridization is superior for the isolation of rare mRNAs; however, its utility is somewhat limited by the fact that large amounts of driver mRNA are required for the procedure. We have used the polymerase chain reaction (PCR) to amplify minute quantities of unknown subtracted sequences, and thus, enhanced the sensitivity and ease of cloning differentially expressed mRNAs. We demonstrate the utility of this technique by describing the isolation of at least four unique cDNA clones of human endothelial cell mRNAs that are induced by the tumor promoter, phorbol ester, a factor that promotes immediate early events during the endothelial cell differentiation pathway in vitro.
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Laboratory of Molecular Biology, Jerome Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, Maryland 20855.
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D001483:Base Sequence; D015152:Blotting, Northern; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000235:genetics*; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D004247:DNA; D003513:Cycloheximide; D013755:Tetradecanoylphorbol Acetate