Analysis of Early Gene Responses

Ronald S. Duman1

1 Yale University School of Medicine and Connecticut Mental Health Center, New Haven, Connecticut
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 2.5
DOI:  10.1002/0471141755.ph0205s00
Online Posting Date:  May, 2001
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Abstract

Early response genes, also referred to as immediate early genes, are rapidly induced in response to a variety of extracellular stimuli, and have been used as markers of cellular activation. Expression of immediate early genes, such as c‐fos, can be used to determine the influence of specific drugs on receptor regulation of intracellular signaling pathways. This unit describes two techniques for the analysis of mRNA from immediate early genes. Northern blot analysis provides a method for size‐fractionation of native mRNA by gel electrophoresis and visualization of a specific mRNA in a pool of total RNA isolated from a tissue of interest. Thus, this analysis provides important information on the size and abundance of the mRNA of interest and on the specificity of the hybridization probe. However, this approach does not provide information on the cellular localization of the mRNA. Such data may be obtained by in situ hybridization of mRNA in tissue sections. In addition to the northern and in situ hybridization techniques, this unit provides two support protocols for radiolabeling either RNA or DNA probes along with information on the selection of an appropriate probe and hybridization conditions.

     
 
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Table of Contents

  • Basic Protocol 1: Northern Blot Analysis of Early Gene Responses
  • Basic Protocol 2: In Situ Hybridization Analysis of Early Gene Responses
  • Support Protocol 1: Labeling of Riboprobes
  • Support Protocol 2: Random‐Primed Labeling of DNA Probes
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Northern Blot Analysis of Early Gene Responses

  Materials
  • Tissue or cells of interest
  • Guanidine isothiocyanate (GIT) buffer (see recipe), ice cold
  • 5.7 M CsCl solution (see recipe)
  • DEPC‐treated H 2O (see recipe)
  • 3 M sodium acetate, pH 5.2, DEPC‐treated (see recipe)
  • 70% and 100% ethanol, ice cold (prepare 70% with recipeDEPC‐treated H 2O )
  • Agarose, RNase‐free (e.g., SeaKem LE from FMC Bioproducts)
  • 20× MOPS buffer (see recipe)
  • 10 mg/ml ethidium bromide (commercially available stock or see appendix 2A)
  • 37% formaldehyde solution (commercially available)
  • Sample loading buffer (see recipe)
  • 20× SSC (see recipe)
  • 22 µm nitrocellulose membrane (e.g., Nitropure from Micron Separation Systems, available through Fisher Scientific)
  • Sheared salmon sperm DNA (e.g., Sigma)
  • Northern hybridization buffer (see recipe)
  • Radiolabeled probe solution (see Support Protocols protocol 31 or protocol 42)
  • 10% (w/v) SDS (prepare with recipeDEPC‐treated H 2O )
  • Whatman 3MM filter paper (≥30‐cm‐long sheets)
  • Absorbent filters (11 × 14–cm sheets; e.g., Life Technologies) or paper towels
  • 50‐ml centrifuge tubes
  • Polytron tissue homogenizer (Brinkmann)
  • 10‐ml ultracentrifuge tubes
  • RNase‐free microcentrifuge tubes (see )
  • Water bath, adjustable between 22° and 100°C
  • Horizontal gel electrophoresis tank with casting tray and combs
  • Transfer apparatus: platform and glass or plastic plate that are at least the size of the gel; reservoir dish larger than the platform and sufficiently deep to hold the transfer buffer
  • 16.5 × 20.3–cm heat‐sealable pouch (for hybridization in a water bath)
  • Hybridization oven (optional)

Basic Protocol 2: In Situ Hybridization Analysis of Early Gene Responses

  Materials
  • Frozen tissue block (ready for cryostat sectioning)
  • 4% paraformaldehyde (see recipe)
  • PBS: 10 mM sodium phosphate/140 mM NaCl, pH 7.4, prepared in DEPC‐treated H 2O (see recipe for DEPC‐treated water)
  • 0.1 M triethanolamine⋅Cl (prepare in DEPC‐treated H 2O—see recipe; adjust pH to 8.0 with glacial acetic acid; store up to 1 yr at room temperature)
  • 0.25% acetic anhydride/0.1 M triethanolamine⋅Cl (prepare fresh immediately before use)
  • 20× SSC (see recipe)
  • 30%, 70%, and 100% ethanol
  • In situ hybridization buffer (see recipe)
  • Radiolabeled riboprobe (see protocol 3)
  • Hybridization chamber buffer ( recipe4× SSC/50% formamide in DEPC‐treated H 2O; see recipe for DEPC‐treated water)
  • RNase digestion buffer (see recipe) with and without 20 µg/ml RNase A (e.g., Sigma)
  • Cryostat
  • Glass staining dishes (e.g., Wheaton dishes from Fisher Scientific) for 20 slides, with removable racks
  • Incubator or water bath at 55°C
  • Hybridization chamber (e.g., Nalgene utility box with lid)

Support Protocol 1: Labeling of Riboprobes

  Materials
  • Miniprep of plasmid template DNA
  • Restriction enzyme and appropriate buffer for complete linearization of plasmid DNA
  • DEPC‐treated H 2O (see recipe)
  • 10% (w/v) SDS (prepare with recipeDEPC‐treated H 2O )
  • 2 mg/ml proteinase K (e.g., Boehringer Mannheim)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (made with buffered phenol—see recipe)
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 3 M sodium acetate, pH 6.0, DEPC‐treated (see recipe)
  • 70% and 100% ethanol, ice cold
  • RNA transcription kit (e.g., Stratagene or equivalent; store all solutions at −20°C), containing:
  •  5× transcription buffer
  •  0.75 M DTT
  •  10 mM premixed nucleotides (choose three from 10 mM solutions of ATP, GTP, CTP, UTP, excluding the nucleotide that will be used to radiolabel the probe; premix at 1:1:1, yielding 3.3 mM each nucleotide)
  •  RNase block
  •  T3, T7 and/or SP6 RNA polymerases
  •  10 U/µl RNase‐free DNase
  • Radiolabeled nucleotide (e.g., NEN Life Sciences): [α‐32P]NTP (800 Ci/mmol; 10 mCi/ml) for northern blot or [α‐35S]NTP (1250 Ci/mmol; 12.5 mCi/ml) for in situ hybridization
  • 0.5 M EDTA, pH 8.0 ( appendix 2A), DEPC‐treated as for DEPC‐treated H 2O (see recipe)
  • STE (see recipe)
  • NUCTRAP push column (Stratagene)
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, )

Support Protocol 2: Random‐Primed Labeling of DNA Probes

  Materials
  • Gel‐purified template DNA
  • Random‐primed labeling kit (e.g., Life Technologies or equivalent), containing:
  •  500 mM dNTPs (dATP, dCTP, dGTP, dTTP)
  •  10× reaction mixture, containing random hexanucleotide primers (54 OD 260 U/ml)
  •  3000 U/ml Klenow fragment of E. coli DNA polymerase I
  • [α‐32P]dNTP (e.g., NEN Life Sciences; 3000 Ci/mmol)
  • 200 mM EDTA (optional)
  • Nick purification column (Pharmacia Biotech) or equivalent
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Figures

Videos

Literature Cited

Literature Cited
   Armstrong, R.C. and Montminy, M.R. 1993. Transynaptic control of gene expression. Annu. Rev. Neurosci. 16:17‐29.
   Brown, T. and Mackey, K. 1997. Analysis of RNA by northern and slot blot hybridization. In Current Protocols in Molecular Biology. (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 4.2.1‐4.2.16. John Wiley & Sons, New York.
   Fields, R.D. 1996. Signaling from neural impulses to genes. Neuroscientist 2:315‐325.
   Gallagher, S.R. 1995. One‐dimensional SDS gel electrophoresis of proteins. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith,and K. Struhl, eds.) pp. 10.2.2‐10.2.35. John Wiley & Sons, New York.
   Gallagher, S., Winston, S.E., Fuller, S.A., and Hurrell, J.G.R. 1997. Immunoblotting and immunodetection. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 10.8.1‐10.8.21. John Wiley & Sons, New York.
   Ghosh, A. and Greenburg, M.E. 1995. Calcium signaling in neurons: Molecular mechanisms and cellular consequences. Science 268:239‐247.
   Metsis, M., Timmusk, T., Arenas, E., Person, H. 1993. Differential usage of multiple brain‐derived neurotrophic factor promoters in the rat brain following neuronal activation. Proc. Natl. Acad. Sci. U.S.A. 90:8802‐8806.
   Morgan, J.I. and Curran, T. 1991. Stimulus‐transcription coupling in the nervous system: Involvement of the inducible proto‐oncogenes fos and jun. Annu. Rev. Neurosci. 14:421‐451.
   Morinobu, S., Strausbaugh, H., Terwilliger, R., and Duman, R.S. 1997. Regulation of c‐Fos and NGF1‐A by antidepressant treatments. Synapse 25:313‐320.
   Murray, K.D., Wood, P.L., Roscasco, C., Isackson, P.J. 1996. A metabotropic glutamate receptor agonist regulates neurotrophin messenger RNA in rat forebrain. Neuroscience 70:617‐630.
   Roberston, L.M., Kerppola, T.K., Vendrell, M., Luk, D., Smeyne, R.J., Bocchiaro, C., Morgan, J.I., and Curran, T. 1995. Regulation of c‐fos expression in transgenic mice requires multiple interdependent transcription control elements. Neuron 14:241‐252.
   Vaidya, V.A., Marek, G.J., Aghajanian, G.K., and Duman, R.S. 1997. 5‐HT2A receptor–mediated regulation of brain‐derived neurotrophic factor mRNA in the hippocampus and neocortex. J. Neurosci. In press.
   Voytas, D. 1997. Agarose gel electrophoresis. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 2.5.1‐2.5.9. John Wiley & Sons, New York.
   Watkins, S. 1989a. Cryosectioning. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 14.2.1‐14.2.8. John Wiley & Sons, New York.
   Watkins, S. 1989b. Immunohistochemistry. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 14.6.1‐14.6.13. John Wiley & Sons, New York.
   Zafra, F., Lindholm, D., Castren, E., Hartikka, J., Thoenen, H. 1992. Regulation of brain‐derived neurotrophic factor and nerve growth factor mRNA in primary cultures of hippocampal neurons and astrocytes. J. Neurosci. 12:4793‐4799.
   Zeller, R. and Rogers, M. 1989. In situ hybridization to cellular RNA. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 14.3.1‐14.3.14. John Wiley & Sons, New York.
Key References
   Bing, G., Filer, D., Miller, J.C., and Stone, E.A. 1991. Noradrenergic activation of immediate early genes in rat cerebral cortex. Mol. Brain Res. 11:43‐46.
  The following is a partial listing of seminal studies demonstrating the regulation of immediate early gene transcription factors by various drugs.
   Cole, A.J., Bhat, R.V., Patt, C., Worley, P.F., and Baraban, J.M. 1992. D1 dopamine receptor activation of multiple transcription factor genes in rat striatum. J. Neurochem. 58:1420‐1426.
   Deutch, A.Y. and Duman, R.S. 1996. The effects of antipsychotic drugs on Fos protein expression in the prefrontal cortex: Cellular localization and pharmacological characterization. Neuroscience 70:377‐389.
   Duncan, G.E., Johnson, K.B., Breese, G.R. 1993. Topographic patterns of brain activity in response to swim stress: Assessment by 2‐deoxyglucose uptake and expression of fos‐like immunoreactivity. J.Neurosci. 13:3932‐3934.
   Graybiel, A.M., Moratalla, R., Robertson, H.A. 1990. Amphetamine‐ and cocaine‐induced drug‐specific activation of the c‐fos gene in striosome‐matrix compartments and limbic subdivisions of the striatum. Proc.Natl. Acad. Sci. U.S.A. 87:6912‐6916.
   Gubits, R.M., Smith, T.M., Fairhurst, J.L., and Yu, H. 1989. Adrenergic receptors mediate changes in c‐fos mRNA levels in brain. Mol. Brain Res. 6:39‐45.
   Hayward, M.D., Duman, R.S., and Nestler, E.J. 1990. Induction of the c‐fos proto‐oncogene during opiate withdrawal in the locus coeruleus and other regions of the rat brain. Brain Res. 525:265‐266.
   Hope, B., Kosofsky, B., Hyman, S.E., and Nestler, E.J. 1992. Regulation of immediate early gene expression and AP‐1 binding in the rat nucleus accumbens by chronic cocaine. Proc. Natl. Acad. Sci. U.S.A. 89:5764‐5768.
   Leslie, R.A., Moorman, J.M., Coulson, A., and Grahame‐Smith, D.G. 1993. Serotonin 2/1C receptor activation causes a localized expression of the immediate‐early gene c‐fos in rat brain: Evidence for involvement of dorsal raphe nucleus projection fibers. Neuroscience 53:457‐463.
   Merchant, K.M. and Dorsa, D.M. 1993. Differential induction of neurotensin and c‐fos gene expression by typical and atypical antipsychotics. Proc. Natl. Acad. Sci. U.S.A. 90:3447‐3451.
   Moratalla, R., Robertson, H.A., and Graybiel, A.M. 1992. Dynamic regulation of NGFI‐A (sif268, erg1) gene expression in the striatum. J. Neurosci. 12:2609‐2622.
   Morgan and Curran, 1991. See above.
   Morgan, J.I., Cohen, D.R., Hempstead, J.L., Curran, T. 1987. Mapping patterns of c‐fos expression in the central nervous system after seizure. Science 237:192‐197.
   Morinobu, S., Nibuya, M., and Duman, R.S. 1995. Chronic antidepressant treatment down‐regulates the induction of c‐fos mRNA in response to acute stress in rat frontal cortex. Neuropsychopharmacology 12:221‐228.2.
   Morinobu et al., 1997. See above.
   Nguyen, T.V., Kosofsky, B.E., Birnbaum, R., Cohen, B.M., and Hyman, S.E. 1992. Differential expression of c‐Fos and Zif268 in rat striatum after haloperidol, clozapine, and amphetamine. Proc. Natl. Acad. Sci. U.S.A. 89:4270‐4274.
   Nibuya, M., Nestler, E.J., and Duman, R.S. 1996. Chronic antidepressant treatment increases the expression of CREB and BDNF in rat hippocampus. J. Neurosci. 16:2365‐2372.
   Sagar, S.M., Sharp, F.R., Curran, T. 1988. Expression of c‐fos protein in brain: Metabolic mapping at the cellular level. Science 240:1328‐1331.
   Young, S.T., Porrino, L.J., and Iadarola, M.J. 1991. Cocaine induces striatal c‐Fos‐immunoreactive proteins via dopaminergic D1 receptors. Proc. Natl. Acad. Sci. U.S.A. 88:1291‐1295.
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