Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Dec;86(23):9139–9143. doi: 10.1073/pnas.86.23.9139

Specific gene suppression by engineered ribozymes in monkey cells.

F H Cameron 1, P A Jennings 1
PMCID: PMC298449  PMID: 2556702

Abstract

Short catalytic RNAs possessing specific endoribonuclease activity (ribozymes) have recently been designed that can potentially shear any chosen target RNA in trans at a specific site. Here, engineered ribozymes targeted against chloramphenicol acetyltransferase (CAT), derived from Tn9, have been cloned into a mammalian expression vector and tested in transient transfection experiments for their effects on CAT expression in monkey (COS1) cells. The ribozymes contained the catalytic domain of the satellite RNA from tobacco ringspot virus and were targeted to three sites in the CAT mRNA by flanking antisense sequences. These ribozymes, which were previously shown to accurately cleave CAT message in vitro, were cloned into a replicating plasmid vector under the control of the highly active simian virus 40 early promoter. The ribozyme gene sequence was incorporated into the 3' untranslated region of the gene for firefly luciferase as it was ineffective when expressed as a short RNA. Each ribozyme construction gave a similar level of suppression of CAT activity when the target was transcribed from the herpes virus thymidine kinase promoter. One of the three (ribozyme 2) was chosen for further study and tested after it had been modified by the addition of extra flanking bases. The reporter gene for luciferase was used to monitor ribozyme level and to function as a specificity control, and the human growth hormone gene was cotransfected as an independent reporter for specificity of the ribozyme against the intended target CAT. At high (approximately 1000-fold) molar excess this ribozyme was demonstrated to consistently and specifically suppress CAT expression (up to approximately 60%) in COS1 cells relative both to a plasmid clone with the ribozyme inserted in the reversed (inactive) orientation and to a control corresponding to the relevant 26-nucleotide antisense segment of CAT.

Full text

PDF

Selected References

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

  1. Buzayan J. M., Gerlach W. L., Bruening G. Satellite tobacco ringspot virus RNA: A subset of the RNA sequence is sufficient for autolytic processing. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8859–8862. doi: 10.1073/pnas.83.23.8859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chang L. J., Stoltzfus C. M. Gene expression from both intronless and intron-containing Rous sarcoma virus clones is specifically inhibited by anti-sense RNA. Mol Cell Biol. 1985 Sep;5(9):2341–2348. doi: 10.1128/mcb.5.9.2341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Coen E. S., Carpenter R. A semi-dominant allele, niv-525, acts in trans to inhibit expression of its wild-type homologue in Antirrhinum majus. EMBO J. 1988 Apr;7(4):877–883. doi: 10.1002/j.1460-2075.1988.tb02891.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  6. Haseloff J., Gerlach W. L. Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature. 1988 Aug 18;334(6183):585–591. doi: 10.1038/334585a0. [DOI] [PubMed] [Google Scholar]
  7. Herskowitz I. Functional inactivation of genes by dominant negative mutations. Nature. 1987 Sep 17;329(6136):219–222. doi: 10.1038/329219a0. [DOI] [PubMed] [Google Scholar]
  8. Holt J. T., Redner R. L., Nienhuis A. W. An oligomer complementary to c-myc mRNA inhibits proliferation of HL-60 promyelocytic cells and induces differentiation. Mol Cell Biol. 1988 Feb;8(2):963–973. doi: 10.1128/mcb.8.2.963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Inouye M. Antisense RNA: its functions and applications in gene regulation--a review. Gene. 1988 Dec 10;72(1-2):25–34. doi: 10.1016/0378-1119(88)90124-2. [DOI] [PubMed] [Google Scholar]
  10. Izant J. G., Weintraub H. Constitutive and conditional suppression of exogenous and endogenous genes by anti-sense RNA. Science. 1985 Jul 26;229(4711):345–352. doi: 10.1126/science.2990048. [DOI] [PubMed] [Google Scholar]
  11. Jennings P. A., Molloy P. L. Inhibition of SV40 replicon function by engineered antisense RNA transcribed by RNA polymerase III. EMBO J. 1987 Oct;6(10):3043–3047. doi: 10.1002/j.1460-2075.1987.tb02610.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Katsuki M., Sato M., Kimura M., Yokoyama M., Kobayashi K., Nomura T. Conversion of normal behavior to shiverer by myelin basic protein antisense cDNA in transgenic mice. Science. 1988 Jul 29;241(4865):593–595. doi: 10.1126/science.2456614. [DOI] [PubMed] [Google Scholar]
  13. Kerr S. M., Stark G. R., Kerr I. M. Excess antisense RNA from infectious recombinant SV40 fails to inhibit expression of a transfected, interferon-inducible gene. Eur J Biochem. 1988 Jul 15;175(1):65–73. doi: 10.1111/j.1432-1033.1988.tb14167.x. [DOI] [PubMed] [Google Scholar]
  14. Marcus-Sekura C. J., Woerner A. M., Shinozuka K., Zon G., Quinnan G. V., Jr Comparative inhibition of chloramphenicol acetyltransferase gene expression by antisense oligonucleotide analogues having alkyl phosphotriester, methylphosphonate and phosphorothioate linkages. Nucleic Acids Res. 1987 Jul 24;15(14):5749–5763. doi: 10.1093/nar/15.14.5749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McNeall J., Sánchez A., Gray P. P., Chesterman C. N., Sleigh M. J. Hyperinducible gene expression from a metallothionein promoter containing additional metal-responsive elements. Gene. 1989 Mar 15;76(1):81–88. doi: 10.1016/0378-1119(89)90010-3. [DOI] [PubMed] [Google Scholar]
  16. Melton D. A. Injected anti-sense RNAs specifically block messenger RNA translation in vivo. Proc Natl Acad Sci U S A. 1985 Jan;82(1):144–148. doi: 10.1073/pnas.82.1.144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Miksicek R., Heber A., Schmid W., Danesch U., Posseckert G., Beato M., Schütz G. Glucocorticoid responsiveness of the transcriptional enhancer of Moloney murine sarcoma virus. Cell. 1986 Jul 18;46(2):283–290. doi: 10.1016/0092-8674(86)90745-2. [DOI] [PubMed] [Google Scholar]
  18. Rezaian M. A., Symons R. H. Anti-sense regions in satellite RNA of cucumber mosaic virus form stable complexes with the viral coat protein gene. Nucleic Acids Res. 1986 Apr 25;14(8):3229–3239. doi: 10.1093/nar/14.8.3229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rubenstein J. L., Nicolas J. F., Jacob F. L'ARN non sens (nsARN): un outil pour inactiver spécifiquement l'expression d'un gène donné in vivo. C R Acad Sci III. 1984;299(8):271–274. [PubMed] [Google Scholar]
  20. 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]
  21. Shibahara S., Mukai S., Morisawa H., Nakashima H., Kobayashi S., Yamamoto N. Inhibition of human immunodeficiency virus (HIV-1) replication by synthetic oligo-RNA derivatives. Nucleic Acids Res. 1989 Jan 11;17(1):239–252. doi: 10.1093/nar/17.1.239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sleigh M. J. A nonchromatographic assay for expression of the chloramphenicol acetyltransferase gene in eucaryotic cells. Anal Biochem. 1986 Jul;156(1):251–256. doi: 10.1016/0003-2697(86)90180-6. [DOI] [PubMed] [Google Scholar]
  23. Travali S., Lipson K. E., Jaskulski D., Lauret E., Baserga R. Role of the promoter in the regulation of the thymidine kinase gene. Mol Cell Biol. 1988 Apr;8(4):1551–1557. doi: 10.1128/mcb.8.4.1551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Uhlenbeck O. C. A small catalytic oligoribonucleotide. Nature. 1987 Aug 13;328(6131):596–600. doi: 10.1038/328596a0. [DOI] [PubMed] [Google Scholar]
  25. Zamecnik P. C., Stephenson M. L. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci U S A. 1978 Jan;75(1):280–284. doi: 10.1073/pnas.75.1.280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES