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Use of Antisense Oligonucleotides to Study the Role of CRABPs in Retinoic Acid-Induced Gene Expression

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Retinoid Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 89))

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Abstract

Antisense methodologies have been used extensively to inhibit the expression of specific genes with a view to elucidating their role in particular cellular processes (1). The technique is based on the ability of mRNA to bind, in a sequence-specific fashion, to a complimentary oligonucleotide sequence (the antisense sequence), via Watson-Crick hydrogen bonding. Binding of the oligonucleotide then prevents efficient translation of the mRNA, either by preventing the nbosome from reading the RNA message, or by activation of RNAse H, an enzyme that specifically cleaves the RNA strand of a DNA-RNA duplex (Fig. 1). In either case, the result is the arrest of specific protein synthesis (2). Though primarily used in cell and tissue-culture systems (13), efforts are underway to develop therapeutic applications (4).

Mechanism of action of antisense oligonucleotides. After binding to the complimentary mRNA sequence (1), the oligonucleotides inhibit protein translation by interference with movement of the mRNA through the translation machinery (2), or by inducing RNAse H-dependent degradation of the mRNA in the duplex (3).

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References

  1. Colman, A (1987) Antisense strategies in cell and developmental biology. J Cell Science 97, 399–409

    Google Scholar 

  2. Helene, C (1991) Rational design of sequence-specific oncogene inhibitors based on antisense and antigene oligonucleotides Eur. J Cancer 27, 1466–1471

    Article  PubMed  CAS  Google Scholar 

  3. Krieg, A. M. (1993) Uptake and efficacy of phosphodiester and modified antisense oligonucleotides in primary cell cultures Clin Chem. 39, 710–712

    Google Scholar 

  4. Heidenreich, O., Kang, S.-H, Xu, X, and Nerenberg, M. (1995) Application of antisense technology to therapeutics Mol Med Today 1, 128–133

    Article  PubMed  CAS  Google Scholar 

  5. Crooke, R. M., Graham, M. L., Cooke, M. E., and Crooke, S. T. (1995) In vitro pharmacokinetics of phosphorothioate antisense oligonucleotides J. Pharmacol. Exp Ther. 275, 462–473.

    PubMed  CAS  Google Scholar 

  6. Wagner, R. W (1995) The state of the art in antisense research. Nature Med 1, 1116–1118.

    Article  PubMed  CAS  Google Scholar 

  7. Nugent, P., and Greene, R M. (1995) Antisense oligonucleotides to CRABP I and II alter the expression of TGF-β3, RAR-β, and tenascin in primary cultures of embryonic palate cells In Vitro Cell Develop. Biol. 31, 553–558

    Article  CAS  Google Scholar 

  8. Nugent, P., and Greene, R. M (1994) Interactions between the TGF-β and retinoic acid signal transduction pathways in embryonic palatal cells. Differentiation 58, 149–155

    Article  PubMed  CAS  Google Scholar 

  9. Nugent, P, Potchinsky, M, Lafferty, C, and Greene, R M (1995) TGF-β modulates the expression of retinoic acid-induced RAR-β expression in primary cultures of embryonic palate cells. Exp. Cell Res. 220, 495–500.

    Article  PubMed  CAS  Google Scholar 

  10. Stoner, C M and Gudas, L J. (1989) Mouse cellular retinoic acid binding protein: cloning, complimentary DNA sequence, and messenger RNA expression during retinoic acid-induced differentiation of F9 wild type and RA-3-10 mutant teratocarcinoma cells Cancer Res 49, 1497–1504

    PubMed  CAS  Google Scholar 

  11. Giguere, V., Lyn, S., Yip, P., Siu, C-H., and Amin, S. (1990) Molecular cloning of cDNA encoding a second cellular retinoic acid-binding protein. Proc. Natl Acad. Sci. USA 87. 6233–6237

    Article  PubMed  CAS  Google Scholar 

  12. Miller, D. A, Lee, A., Matsui, Y, Chen, E Y, Moses, H L., and Derynck, R (1989) Complementary DNA cloning of the murine transforming growth factor-β3 (TGFβ3) precursor and the comparative expression of TGFβ3 and TGFβl messenger RNA in murine embryos and adult tissues. Mol Endocrinol 3, 1926–1934

    Article  PubMed  CAS  Google Scholar 

  13. Zelent, A, Krust, A., Petkovich, M., Kastner, P., and Chambon, P (1989) Cloning of murine α and β retinoic acid receptors and a novel receptor γ predominantly expressed in skin. Nature 339, 714–717

    Article  PubMed  CAS  Google Scholar 

  14. Saga, Y., Tsukamoto, T, Jing, N., Kusakabe, M., and Sakakura, T (1991) Murine tenascin cDNA cloning, structure and temporal expression of isoforms. Gene 104, 177–185.

    Article  PubMed  CAS  Google Scholar 

  15. Sambrook, J, Fritsch, E F, and Maniatis, T (1989) Extraction, purification, and analysis of messenger RNA from eukaryotic cells, in Molecular Cloning A Laboratory Manual, 2nd ed. (Ford, N., ed.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 7 1–7 87

    Google Scholar 

  16. Krumlauf, R. (1991) Northern blot analysis of gene expression, in Methods in Molecular Biology, Vol. 7. Gene Transfer and Expression Protocols (Murray,E J, ed.), Humana,Totowa, NJ, pp. 307–323

    Chapter  Google Scholar 

  17. Chomczynski, P and Saachi, N (1987) Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.

    Article  PubMed  CAS  Google Scholar 

  18. Siegenthaler, G. (1990) Gel electrophoresis of cellular retinoic acid-binding protein, cellular retinol-binding protein, and serum retinol-binding protein. Meth. Enzymol 189, 299–307

    Article  PubMed  CAS  Google Scholar 

  19. Johnson, B., McClain, S. G., Doran, E R., Tice, G, and Kirsch, M A (1990) Rapid purification of synthetic oligonucleotides, a convenient alternative to HPLC and polyacrylamide gel electrophoresis. Biotechniques 8, 424–429

    PubMed  CAS  Google Scholar 

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© 1998 Humana Press Inc, Totowa, NJ

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Nugent, P., Greene, R.M. (1998). Use of Antisense Oligonucleotides to Study the Role of CRABPs in Retinoic Acid-Induced Gene Expression. In: Redfern, C.P.F. (eds) Retinoid Protocols. Methods in Molecular Biology, vol 89. Humana Press. https://doi.org/10.1385/0-89603-438-0:191

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  • DOI: https://doi.org/10.1385/0-89603-438-0:191

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-438-9

  • Online ISBN: 978-1-59259-573-0

  • eBook Packages: Springer Protocols

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