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. 1996 Jul 15;24(14):2697–2700. doi: 10.1093/nar/24.14.2697

High-titer bicistronic retroviral vectors employing foot-and-mouth disease virus internal ribosome entry site.

N Ramesh 1, S T Kim 1, M Q Wei 1, M Khalighi 1, W R Osborne 1
PMCID: PMC146003  PMID: 8758998

Abstract

Bicistronic retroviral vectors were constructed containing the foot-and-mouth disease virus (FMDV) internal ribosome entry site (IRES) followed by the coding region of beta-galactosidase (beta-gal) or therapeutic genes, with the selectable neomycin phosphotransferase gene under the control of the viral long terminal repeat (LTR) promoter. LNFX, a vector with a multiple cloning site 3' to foot-and-mouth disease virus IRES, was used to construct vectors encoding rat erythropoietin (EP), rat granulocyte colony-stimulating factor (G-CSF), human adenosine deaminase (ADA) and beta-gal. In transduced primary rat vascular smooth muscle cells the cytokines were expressed at high levels, similar to those obtained from vectors employing the viral LTR promoter. LNFZ, a vector encoding beta-gal, had a 10-fold increase in titer over that of LNPoZ, a comparable vector containing the poliovirus (Po) internal ribosome entry site. Primary canine vascular smooth muscle cells infected with LNFZ and LNPoZ expressed similar activities of beta-gal and neomycin phosphotransferase (NPT). Overall, these vectors had titers between 10(6) and 2 x 10(7) c.f.u./ml, indicating that foot-and-mouth disease virus IRES provides high-titer bicistronic vectors with high-level two gene expression.

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Selected References

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  1. Adam M. A., Osborne W. R., Miller A. D. R-region cDNA inserts in retroviral vectors are compatible with virus replication and high-level protein synthesis from the insert. Hum Gene Ther. 1995 Sep;6(9):1169–1176. doi: 10.1089/hum.1995.6.9-1169. [DOI] [PubMed] [Google Scholar]
  2. Adam M. A., Ramesh N., Miller A. D., Osborne W. R. Internal initiation of translation in retroviral vectors carrying picornavirus 5' nontranslated regions. J Virol. 1991 Sep;65(9):4985–4990. doi: 10.1128/jvi.65.9.4985-4990.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Borman A. M., Bailly J. L., Girard M., Kean K. M. Picornavirus internal ribosome entry segments: comparison of translation efficiency and the requirements for optimal internal initiation of translation in vitro. Nucleic Acids Res. 1995 Sep 25;23(18):3656–3663. doi: 10.1093/nar/23.18.3656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dale D. C., Lau S., Nash R., Boone T., Osborne W. Effect of endotoxin on serum granulocyte and granulocyte-macrophage colony-stimulating factor levels in dogs. J Infect Dis. 1992 Apr;165(4):689–694. doi: 10.1093/infdis/165.4.689. [DOI] [PubMed] [Google Scholar]
  5. Ghattas I. R., Sanes J. R., Majors J. E. The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos. Mol Cell Biol. 1991 Dec;11(12):5848–5859. doi: 10.1128/mcb.11.12.5848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hock R. A., Miller A. D., Osborne W. R. Expression of human adenosine deaminase from various strong promoters after gene transfer into human hematopoietic cell lines. Blood. 1989 Aug 1;74(2):876–881. [PubMed] [Google Scholar]
  7. Jang S. K., Kräusslich H. G., Nicklin M. J., Duke G. M., Palmenberg A. C., Wimmer E. A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J Virol. 1988 Aug;62(8):2636–2643. doi: 10.1128/jvi.62.8.2636-2643.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Jang S. K., Wimmer E. Cap-independent translation of encephalomyocarditis virus RNA: structural elements of the internal ribosomal entry site and involvement of a cellular 57-kD RNA-binding protein. Genes Dev. 1990 Sep;4(9):1560–1572. doi: 10.1101/gad.4.9.1560. [DOI] [PubMed] [Google Scholar]
  9. Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. doi: 10.1083/jcb.108.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kühn R., Luz N., Beck E. Functional analysis of the internal translation initiation site of foot-and-mouth disease virus. J Virol. 1990 Oct;64(10):4625–4631. doi: 10.1128/jvi.64.10.4625-4631.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lim K., Chae C. B. A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for beta-galactosidase. Biotechniques. 1989 Jun;7(6):576–579. [PubMed] [Google Scholar]
  12. Martínez-Salas E., Sáiz J. C., Dávila M., Belsham G. J., Domingo E. A single nucleotide substitution in the internal ribosome entry site of foot-and-mouth disease virus leads to enhanced cap-independent translation in vivo. J Virol. 1993 Jul;67(7):3748–3755. doi: 10.1128/jvi.67.7.3748-3755.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Miller A. D., Rosman G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980-2, 984-6, 989-90. [PMC free article] [PubMed] [Google Scholar]
  14. Morgan R. A., Couture L., Elroy-Stein O., Ragheb J., Moss B., Anderson W. F. Retroviral vectors containing putative internal ribosome entry sites: development of a polycistronic gene transfer system and applications to human gene therapy. Nucleic Acids Res. 1992 Mar 25;20(6):1293–1299. doi: 10.1093/nar/20.6.1293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mountford P. S., Smith A. G. Internal ribosome entry sites and dicistronic RNAs in mammalian transgenesis. Trends Genet. 1995 May;11(5):179–184. doi: 10.1016/S0168-9525(00)89040-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Oh S. K., Sarnow P. Gene regulation: translational initiation by internal ribosome binding. Curr Opin Genet Dev. 1993 Apr;3(2):295–300. doi: 10.1016/0959-437X(93)90037-P. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Osborne W. R., Miller A. D. Design of vectors for efficient expression of human purine nucleoside phosphorylase in skin fibroblasts from enzyme-deficient humans. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6851–6855. doi: 10.1073/pnas.85.18.6851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Osborne W. R., Ramesh N., Lau S., Clowes M. M., Dale D. C., Clowes A. W. Gene therapy for long-term expression of erythropoietin in rats. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):8055–8058. doi: 10.1073/pnas.92.17.8055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pelletier J., Kaplan G., Racaniello V. R., Sonenberg N. Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5' noncoding region. Mol Cell Biol. 1988 Mar;8(3):1103–1112. doi: 10.1128/mcb.8.3.1103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pilipenko E. V., Blinov V. M., Chernov B. K., Dmitrieva T. M., Agol V. I. Conservation of the secondary structure elements of the 5'-untranslated region of cardio- and aphthovirus RNAs. Nucleic Acids Res. 1989 Jul 25;17(14):5701–5711. doi: 10.1093/nar/17.14.5701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pilipenko E. V., Blinov V. M., Romanova L. I., Sinyakov A. N., Maslova S. V., Agol V. I. Conserved structural domains in the 5'-untranslated region of picornaviral genomes: an analysis of the segment controlling translation and neurovirulence. Virology. 1989 Feb;168(2):201–209. doi: 10.1016/0042-6822(89)90259-6. [DOI] [PubMed] [Google Scholar]
  22. Ramesh N., Osborne W. R. Assay of neomycin phosphotransferase activity in cell extracts. Anal Biochem. 1991 Mar 2;193(2):316–318. doi: 10.1016/0003-2697(91)90028-r. [DOI] [PubMed] [Google Scholar]
  23. Sonenberg N. Poliovirus translation. Curr Top Microbiol Immunol. 1990;161:23–47. doi: 10.1007/978-3-642-75602-3_2. [DOI] [PubMed] [Google Scholar]
  24. Spivak J. L., Pham T., Isaacs M., Hankins W. D. Erythropoietin is both a mitogen and a survival factor. Blood. 1991 Mar 15;77(6):1228–1233. [PubMed] [Google Scholar]
  25. Trono D., Pelletier J., Sonenberg N., Baltimore D. Translation in mammalian cells of a gene linked to the poliovirus 5' noncoding region. Science. 1988 Jul 22;241(4864):445–448. doi: 10.1126/science.2839901. [DOI] [PubMed] [Google Scholar]
  26. Wen D., Boissel J. P., Tracy T. E., Gruninger R. H., Mulcahy L. S., Czelusniak J., Goodman M., Bunn H. F. Erythropoietin structure-function relationships: high degree of sequence homology among mammals. Blood. 1993 Sep 1;82(5):1507–1516. [PubMed] [Google Scholar]

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