Expression of phosphoenolpyruvate carboxykinase (PEPCK) chimeras in renal epithelial cells. Retention of appropriate physiological responsiveness using enhancerless retroviral vectors.
We used an enhancerless U3 mutant retroviral vector to deliver chimeras of the phosphoenolpyruvate carboxykinase (PEPCK) promoter region to a renal epithelial cell line capable of expressing PEPCK mRNA. Chimeras consisting of the PEPCK promoter and chloramphenicol acetyltransferase, neomycin phosphotransferase or human growth hormone genes were expressed after viral infection of the NRK52E renal epithelial cell line. Virus-delivered sequences in which the direction of PEPCK promoter transcription was antegrade to the normal direction of the long terminal repeat (LTR)-initiated transcription correctly upon stimulation with dexamethasone or 8-bromo cyclic AMP and upon lowering of the extracellular pH. Fluorescent primer extension in situ using primers specific for virus-delivered sequences of antegrade constructs indicated that a large fraction of NRK52E cells could be infected by co-cultivation with virus-producing psi-2 cells without G418 selection. Virus-delivered constructs whose orientation was opposite to that of the LTRs were expressed at very low levels, with transcripts detectable by PCR only in RNA from cyclic AMP-treated cells. Using reverse transcription/PCR, we demonstrated that the chimeric transcripts were from the internal PEPCK promoter rather than a functional or reconstituted Moloney LTR. PEPCK-reporter chimeras delivered by retroviral vectors demonstrated a level of expression more consistent with the level of expression of the native PEPCK gene than did transfected chimeras. This expression system should prove useful for studies of the physiological modulation of gene expression in renal tissues.
Pollock
A S
AS
University of California, San Francisco.
Lovett
D H
DH
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002678:Chimera; D004742:Enhancer Elements, Genetic; D004848:Epithelium; D005822:Genetic Vectors; D019868:Kanamycin Kinase; D007668:Kidney; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics*; D009838:Oligodeoxyribonucleotides; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000096:biosynthesis / Q000235:genetics*; D010770:Phosphotransferases / Q000096:biosynthesis / Q000235:genetics*; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D011401:Promoter Regions, Genetic; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D014158:Transcription, Genetic
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011061:Poly A; D012313:RNA; D010770:Phosphotransferases; D019868:Kanamycin Kinase; D010729:Phosphoenolpyruvate Carboxykinase (GTP)