Advertisement

Antisense RNA/DNA-Based Techniques to Probe Adrenergic Receptor Function

  • Hsien-Yu Wang
  • Fubao Lin
  • Craig C. Malbon
Part of the Methods in Molecular Biology™ book series (MIMB, volume 126)

Abstract

Ablation of the mRNA of a targeted protein by the use of antisense DNA and RNA provides degrees of freedom not available in many other strategies to suppress or eliminate gene products (1, 2, 3). Numerous examples exist demonstrating the utility of the antisense DNA/RNA strategy for study of signaling (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18). In the case of ODNs, preparation of reagents requires no additional skill other than knowing the commercial supplier for ODN synthesis and purification (19). Expression of antisense RNA requires a scientific facility exhibiting simple techniques of molecular biology and can be accomplished by a variety of approaches, including constitutive expression by a strong promoter; this latter approach requires no regulation and assumes functional compatibility with the targeted cells (4,9,11,12). Promoters that can be “induced” afford an additional capability; expression of antisense RNA being turned “on” and again “off” in response to molecular signals provide approaches to RNA induction or suppression. The inducibility of antisense RNA is of particular utility in the suppression of mRNAs that encode proteins necessary for viability in cells or in the whole animal. Traditional “knockout” of genes by homologous recombination that are crucial targets leads to lethality in the transgenic mice system and consequently no viable pups. Inducible antisense RNA transgenes are maintained “silently” in utero and later can be turned “on” at birth or thereafter, permitting production of viable transgenic pups. In the “technical knockouts” (TKOs) rendered by inducible antisense RNA vectors, the additional time and expense of breeding to homozygosity in traditional knockouts is avoided, the output of antisense RNA product from a single transgene being sufficient to silence the mRNA for most protein targets.

Keywords

Antisense Sequence Antisense ODNs Target Gene Product Inhibitory Adenylylcyclase PEPCK Gene 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Miller, P. S., Braiterman, L. T., and Ts’o, P. O. (1977) Effects of a trinucleotide ethyl phosphotriester, Gmp(Et)Gmp(Et)U, on mammalian cells in culture. Biochemistry 16, 1988–1996.PubMedCrossRefGoogle Scholar
  2. 2.
    Haseloff, J. and Gerlach, W. L. (1988) Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature 334, 585–591.PubMedCrossRefGoogle Scholar
  3. 3.
    Goodchild, J. (1989) Oligodeoxynucleotides-antisense inhibitors of gene expression. (Cohen, C. H., ed.), CRC, Boca Raton, FL, pp. 53–77.Google Scholar
  4. 4.
    Bahouth, S. W., Park, E. A., Beauchamp, M., Cui, X., and Malbon, C. C. (1996) Identification of a glucocorticoid repressor domain in the rat beta 1-adrenergic receptor gene. Receptors Signal Transduc. 6, 141–149.Google Scholar
  5. 5.
    Moxham, C. M., Hod, Y., and Malbon, C. C. (1993) Gi alpha 2 mediates the inhibitory regulation of adenylylcyclase in vivo: analysis in transgenic mice with Gi alpha 2 suppressed by inducible antisense RNA. Dev. Genet. 14, 266–273.PubMedCrossRefGoogle Scholar
  6. 6.
    Moxham, C. M., Hod, Y., and Malbon, C. C. (1993) Induction of G alpha i2-specific antisense RNA in vivo inhibits neonatal growth. Science 260, 991–995.PubMedCrossRefGoogle Scholar
  7. 7.
    Moxham, C. M. and Malbon, C. C. (1996) Insulin action impaired by deficiency of the G-protein subunit Gi alpha2. Nature 379, 840–844.PubMedCrossRefGoogle Scholar
  8. 8.
    Shih, M. and Malbon, C. C. (1994) Oligodeoxynucleotides antisense to mRNA encoding protein kinase A, protein kinase C, and beta-adrenergic receptor kinase reveal distinctive cell-type-specific roles in agonist-induced desensitization. Proc. Nat. Acad. Sci. USA 91, 12,193–12,197.PubMedCrossRefGoogle Scholar
  9. 9.
    Shih, M. and Malbon, C. C. (1996) Protein kinase C deficiency blocks recovery from agonist-induced desensitization. J. Biol. Chem. 271, 21,478–21,483.PubMedCrossRefGoogle Scholar
  10. 10.
    Wang, H. Y., Watkins, D. C., and Malbon, C. C. (1992) Antisense oligodeoxy-nucleotides to Gs protein alpha-subunit sequence accelerate differentiation of fibroblasts to adipocytes. Nature 358, 334–337.PubMedCrossRefGoogle Scholar
  11. 11.
    Watkins, D. C., Johnson, G. L., and Malbon, C. C. (1992) Regulation of the differentiation of teratocarcinoma cells into primitive endoderm by G alpha i2. Science 258, 1373–1375.PubMedCrossRefGoogle Scholar
  12. 12.
    Watkins, D. C., Moxham, C. M., Morris, A. J., and Malbon, C. C. (1994) Suppression of Gi alpha 2 enhances phospholipase C signalling. Biochem. J. 299, 593–596.PubMedGoogle Scholar
  13. 13.
    Dean, N. and McKay, R. (1995) Inhibition of protein kinase C-alpha expression in mice after systemic administration of phosphorothioate antisense oligodeoxynucleotides. Proc. Nat. Acad. Sci. USA 91, 11,762–11,766.CrossRefGoogle Scholar
  14. 14.
    Shih, M., Lin, F., Scott, J. D., Wang, H.-Y., and Malben, C. C. (1999) Dynamic complexation of β2-adrenergic receptors with protein kinases and phosphatases. J. Biol. Chem. 274, 1588–1595.PubMedCrossRefGoogle Scholar
  15. 15.
    Kleuss, C., Hescheler, J., Ewel, C., Rosenthal, W., Schultz, G., and Wittig, B. (1991) Assignment of G-protein subtypes to specific receptors inducing inhibition of calcium currents. Nature 353, 43–48.PubMedCrossRefGoogle Scholar
  16. 16.
    Kleuss, C., Scherubl, H., Hescheler, J., Schultz, G., and Wittig, B. (1992) Different beta-subunits determine G-protein interaction with transmembrane receptors [see comments]. Nature 358, 424–426.PubMedCrossRefGoogle Scholar
  17. 17.
    Kleuss, C., Scherubl, H., Hescheler, J., Schultz, G., and Wittig, B. (1993) Selectivity in signal transduction determined by gamma subunits of heterotrimeric G proteins. Science 259, 832–834.PubMedCrossRefGoogle Scholar
  18. 18.
    Kleuss, C., Schultz, G., and Wittig, B. (1994) Microinjection of antisense oligonucleotides to assess G-protein subunit function. Methods Enzymol. 237, 345–355.PubMedCrossRefGoogle Scholar
  19. 19.
    Wagner, R. W. (1994) Gene inhibition using antisense oligodeoxynucleotides. (review) (53 refs). Nature 372, 333–335.PubMedCrossRefGoogle Scholar
  20. 20.
    Sauer, B. (1993) Manipulation of transgenes by site-specific recombination: use of Cre recombinase. Methods Enzymol. 225, 890–900.PubMedCrossRefGoogle Scholar
  21. 21.
    Katsuki, H., Kaneko, S., and Satoh, M. (1992) Involvement of postsynaptic G proteins in hippocampal long-term potentiation. Brain Research 581, 108–114.PubMedCrossRefGoogle Scholar
  22. 22.
    Agrawal, S. (1996) Methods in Molecular Medicine, vol. 1: Antisense Therapeutics. Humana, Totowa, NJ.Google Scholar
  23. 23.
    Gollasch, M., Kleuss, C., Hescheler, J., Wittig, B., and Schultz, G. (1993) Gi2 and protein kinase C are required for thyrotropin-releasing hormone-induced stimulation of voltage-dependent Ca2+ channels in rat pituitary GH3 cells. Proc. Natl. Acad. Sci. USA 90, 6265–6269.PubMedCrossRefGoogle Scholar
  24. 24.
    Kleuss, C., Raw, A. S., Lee, E., Sprang, S. R., and Gilman, A.G. (1994) Mechanism of GTP hydrolysis by G-protein alpha subunits. Proc. Nat. Acad. Sci. USA 91, 9828–9831.PubMedCrossRefGoogle Scholar
  25. 25.
    Galvin-Parton, P. A., Chen, X., Moxham, C. M., and Malbon, C. C. (1997) Induction of Galphaq-specific antisense RNA in vivo causes increased body mass and hyperadiposity. J. Biol. Chem. 272, 4335–4341.PubMedCrossRefGoogle Scholar
  26. 26.
    Guo, J. H., Wang, H. Y., and Malbon, C. C. (1998) Conditional, tissue-specific expression of Q205L G-alpha12 in vivo mimics insulin activation of Jun N-terminal kinase and P38 kinase. J. Biol. Chem. 273, 16,487–16,493.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Hsien-Yu Wang
    • 1
  • Fubao Lin
    • 2
  • Craig C. Malbon
    • 2
  1. 1.Department of Physiology and Biophysics, Diabetes and Metabolic Diseases Research CenterSchool of Medicine, State University of New YorkStony Brook
  2. 2.Department of Molecular Pharmacology-HSC, Diabetes and Metabolic Diseases Research CenterSchool of Medicine, State University of New YorkStony Brook

Personalised recommendations