Antisense RNA/DNA-Based Techniques to Probe Adrenergic Receptor Function
Part of the
Methods in Molecular Biology™
book series (MIMB, volume 126)
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.
KeywordsAntisense 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.
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
, 1988–1996.PubMedCrossRefGoogle Scholar
Haseloff, J. and Gerlach, W. L. (1988) Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature
, 585–591.PubMedCrossRefGoogle Scholar
Goodchild, J. (1989) Oligodeoxynucleotides-antisense inhibitors of gene expression. (Cohen, C. H., ed.), CRC, Boca Raton, FL, pp. 53–77.Google Scholar
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.
, 141–149.Google Scholar
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.
, 266–273.PubMedCrossRefGoogle Scholar
Moxham, C. M., Hod, Y., and Malbon, C. C. (1993) Induction of G alpha i2-specific antisense RNA in vivo inhibits neonatal growth. Science
, 991–995.PubMedCrossRefGoogle Scholar
Moxham, C. M. and Malbon, C. C. (1996) Insulin action impaired by deficiency of the G-protein subunit Gi alpha2. Nature
, 840–844.PubMedCrossRefGoogle Scholar
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
, 12,193–12,197.PubMedCrossRefGoogle Scholar
Shih, M. and Malbon, C. C. (1996) Protein kinase C deficiency blocks recovery from agonist-induced desensitization. J. Biol. Chem.
, 21,478–21,483.PubMedCrossRefGoogle Scholar
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
, 334–337.PubMedCrossRefGoogle Scholar
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
, 1373–1375.PubMedCrossRefGoogle Scholar
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.
, 593–596.PubMedGoogle Scholar
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
, 11,762–11,766.CrossRefGoogle Scholar
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.
, 1588–1595.PubMedCrossRefGoogle Scholar
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
, 43–48.PubMedCrossRefGoogle Scholar
Kleuss, C., Scherubl, H., Hescheler, J., Schultz, G., and Wittig, B. (1992) Different beta-subunits determine G-protein interaction with transmembrane receptors [see
, 424–426.PubMedCrossRefGoogle Scholar
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
, 832–834.PubMedCrossRefGoogle Scholar
Kleuss, C., Schultz, G., and Wittig, B. (1994) Microinjection of antisense oligonucleotides to assess G-protein subunit function. Methods Enzymol.
, 345–355.PubMedCrossRefGoogle Scholar
Wagner, R. W. (1994) Gene inhibition using antisense oligodeoxynucleotides. (review) (53 refs). Nature
, 333–335.PubMedCrossRefGoogle Scholar
Sauer, B. (1993) Manipulation of transgenes by site-specific recombination: use of Cre recombinase. Methods Enzymol.
, 890–900.PubMedCrossRefGoogle Scholar
Katsuki, H., Kaneko, S., and Satoh, M. (1992) Involvement of postsynaptic G proteins in hippocampal long-term potentiation. Brain Research
, 108–114.PubMedCrossRefGoogle Scholar
Agrawal, S. (1996) Methods in Molecular Medicine, vol. 1: Antisense Therapeutics
. Humana, Totowa, NJ.Google Scholar
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
, 6265–6269.PubMedCrossRefGoogle Scholar
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
, 9828–9831.PubMedCrossRefGoogle Scholar
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.
, 4335–4341.PubMedCrossRefGoogle Scholar
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.
, 16,487–16,493.PubMedCrossRefGoogle Scholar
© Humana Press Inc., Totowa, NJ 2000