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. 1992 Jun 15;284(Pt 3):725–732. doi: 10.1042/bj2840725

Expression of phosphoenolpyruvate carboxykinase (PEPCK) chimeras in renal epithelial cells. Retention of appropriate physiological responsiveness using enhancerless retroviral vectors.

A S Pollock 1, D H Lovett 1
PMCID: PMC1132599  PMID: 1377912

Abstract

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barklis E., Mulligan R. C., Jaenisch R. Chromosomal position or virus mutation permits retrovirus expression in embryonal carcinoma cells. Cell. 1986 Nov 7;47(3):391–399. doi: 10.1016/0092-8674(86)90596-9. [DOI] [PubMed] [Google Scholar]
  2. Beale E. G., Chrapkiewicz N. B., Scoble H. A., Metz R. J., Quick D. P., Noble R. L., Donelson J. E., Biemann K., Granner D. K. Rat hepatic cytosolic phosphoenolpyruvate carboxykinase (GTP). Structures of the protein, messenger RNA, and gene. J Biol Chem. 1985 Sep 5;260(19):10748–10760. [PubMed] [Google Scholar]
  3. Chrapkiewicz N. B., Beale E. G., Granner D. K. Induction of the messenger ribonucleic acid coding for phosphoenolpyruvate carboxykinase in H4-II-E cells. Evidence for a nuclear effect of cyclic AMP. J Biol Chem. 1982 Dec 10;257(23):14428–14432. [PubMed] [Google Scholar]
  4. Chu G., Hayakawa H., Berg P. Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res. 1987 Feb 11;15(3):1311–1326. doi: 10.1093/nar/15.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cone R. D., Weber-Benarous A., Baorto D., Mulligan R. C. Regulated expression of a complete human beta-globin gene encoded by a transmissible retrovirus vector. Mol Cell Biol. 1987 Feb;7(2):887–897. doi: 10.1128/mcb.7.2.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dass P. D., Wu M. C. Uptake and metabolism of glutamine in cultured kidney cells. Biochim Biophys Acta. 1985 Apr 22;845(1):94–100. doi: 10.1016/0167-4889(85)90059-x. [DOI] [PubMed] [Google Scholar]
  7. Dornburg R., Temin H. M. Retroviral vector system for the study of cDNA gene formation. Mol Cell Biol. 1988 Jun;8(6):2328–2334. doi: 10.1128/mcb.8.6.2328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Engelhardt J. F., Kellum M. J., Bisat F., Pitha P. M. Retrovirus vector-targeted inducible expression of human beta-interferon gene to B-cells. Virology. 1990 Oct;178(2):419–428. doi: 10.1016/0042-6822(90)90339-s. [DOI] [PubMed] [Google Scholar]
  9. Guild B. C., Finer M. H., Housman D. E., Mulligan R. C. Development of retrovirus vectors useful for expressing genes in cultured murine embryonal cells and hematopoietic cells in vivo. J Virol. 1988 Oct;62(10):3795–3801. doi: 10.1128/jvi.62.10.3795-3801.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hatzoglou M., Park E., Wynshaw-Boris A., Kaung H. L., Hanson R. W. Hormonal regulation of chimeric genes containing the phosphoenolpyruvate carboxykinase promoter regulatory region in hepatoma cells infected by murine retroviruses. J Biol Chem. 1988 Nov 25;263(33):17798–17808. [PubMed] [Google Scholar]
  11. Iynedjian P. B., Ballard F. J., Hanson R. W. The regulation of phosphoenolpyruvate carboxykinase (GTP) synthesis in rat kidney cortex. The role of acid-base balance and glucocorticoids. J Biol Chem. 1975 Jul 25;250(14):5596–5603. [PubMed] [Google Scholar]
  12. Klemm D. J., Roesler W. J., Liu J. S., Park E. A., Hanson R. W. In vitro analysis of promoter elements regulating transcription of the phosphoenolpyruvate carboxykinase (GTP) gene. Mol Cell Biol. 1990 Feb;10(2):480–485. doi: 10.1128/mcb.10.2.480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kriegler M., Perez C. F., Hardy C., Botchan M. Transformation mediated by the SV40 T antigens: separation of the overlapping SV40 early genes with a retroviral vector. Cell. 1984 Sep;38(2):483–491. doi: 10.1016/0092-8674(84)90503-8. [DOI] [PubMed] [Google Scholar]
  14. Mann R., Mulligan R. C., Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983 May;33(1):153–159. doi: 10.1016/0092-8674(83)90344-6. [DOI] [PubMed] [Google Scholar]
  15. McGrane M. M., de Vente J., Yun J., Bloom J., Park E., Wynshaw-Boris A., Wagner T., Rottman F. M., Hanson R. W. Tissue-specific expression and dietary regulation of a chimeric phosphoenolpyruvate carboxykinase/bovine growth hormone gene in transgenic mice. J Biol Chem. 1988 Aug 15;263(23):11443–11451. [PubMed] [Google Scholar]
  16. Meisner H., Loose D. S., Hanson R. W. Effect of hormones on transcription of the gene for cytosolic phosphoenolpyruvate carboxykinase (GTP) in rat kidney. Biochemistry. 1985 Jan 15;24(2):421–425. doi: 10.1021/bi00323a027. [DOI] [PubMed] [Google Scholar]
  17. Pollock A. S. Induction of renal phosphoenolpyruvate carboxykinase mRNA: suppressive effect of glucose. Am J Physiol. 1989 Jul;257(1 Pt 2):F145–F151. doi: 10.1152/ajprenal.1989.257.1.F145. [DOI] [PubMed] [Google Scholar]
  18. Pollock A. S., Long J. A. The 5'region of the rat phosphoenolpyruvate carboxykinase gene confers pH sensitivity to chimeric genes expressed in renal and liver cell lines capable of expressing PEPCK. Biochem Biophys Res Commun. 1989 Oct 16;164(1):81–87. doi: 10.1016/0006-291x(89)91685-9. [DOI] [PubMed] [Google Scholar]
  19. Selden R. F., Howie K. B., Rowe M. E., Goodman H. M., Moore D. D. Human growth hormone as a reporter gene in regulation studies employing transient gene expression. Mol Cell Biol. 1986 Sep;6(9):3173–3179. doi: 10.1128/mcb.6.9.3173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Shih C. C., Stoye J. P., Coffin J. M. Highly preferred targets for retrovirus integration. Cell. 1988 May 20;53(4):531–537. doi: 10.1016/0092-8674(88)90569-7. [DOI] [PubMed] [Google Scholar]
  21. Sorge J., Hughes S. H. Splicing of intervening sequences introduced into an infectious retroviral vector. J Mol Appl Genet. 1982;1(6):547–559. [PubMed] [Google Scholar]
  22. Sorge J., Wright D., Erdman V. D., Cutting A. E. Amphotropic retrovirus vector system for human cell gene transfer. Mol Cell Biol. 1984 Sep;4(9):1730–1737. doi: 10.1128/mcb.4.9.1730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Soriano P., Friedrich G., Lawinger P. Promoter interactions in retrovirus vectors introduced into fibroblasts and embryonic stem cells. J Virol. 1991 May;65(5):2314–2319. doi: 10.1128/jvi.65.5.2314-2319.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wynshaw-Boris A., Short J. M., Loose D. S., Hanson R. W. Characterization of the phosphoenolpyruvate carboxykinase (GTP) promoter-regulatory region. I. Multiple hormone regulatory elements and the effects of enhancers. J Biol Chem. 1986 Jul 25;261(21):9714–9720. [PubMed] [Google Scholar]
  25. Yu S. F., von Rüden T., Kantoff P. W., Garber C., Seiberg M., Rüther U., Anderson W. F., Wagner E. F., Gilboa E. Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells. Proc Natl Acad Sci U S A. 1986 May;83(10):3194–3198. doi: 10.1073/pnas.83.10.3194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. de Larco J. E., Todaro G. J. Epithelioid and fibroblastic rat kidney cell clones: epidermal growth factor (EGF) receptors and the effect of mouse sarcoma virus transformation. J Cell Physiol. 1978 Mar;94(3):335–342. doi: 10.1002/jcp.1040940311. [DOI] [PubMed] [Google Scholar]

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