Green Fluorescent Protein in the Study of Neuronal Signaling Pathways

Leslie Blair1, Kendra Bence‐Hanulec1, John Marshall1

1 Brown University, Providence, Rhode Island
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.16
DOI:  10.1002/0471142301.ns0516s14
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

In recent years, techniques have been established for transiently co‐transfecting cells with cDNA of the jellyfish green fluoresent protein (GFP), a reporter gene that encodes a non‐toxic marker. This approach can be applied to primary neurons where it has become especially useful for the study of neuronal second messenger pathways. This unit describes procedures for transfecting neurons in primary culture: transfection with GFP DNA, including co‐transfecting with separate GFP and gene‐of‐interest constructs, transfecting with a single construct containing the gene of interest fused to a GFP gene, and transfecting with a single construct containing separate gene‐of‐interest and GFP cassettes. Also included is a method for the rapid, large‐scale preparation of a nearly homogeneous population of neurons from rat cerebellum. The Commentary provides several examples of how this approach can be applied to specific biological questions on neuronal signaling pathways.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Transfection of Green Fluorescent Protein and Gene‐of‐Interest Constructs
  • Support Protocol 1: Culturing Rat Cerebellar Granule Neurons
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Transfection of Green Fluorescent Protein and Gene‐of‐Interest Constructs

  Materials
  • Rat pup cerebellar granule neurons (see protocol 2)
  • Green fluorescent protein (GFP) cDNA
  • cDNA encoding gene of interest
  • 0.25 M CaCl 2 (sterile), room temperature
  • 2× BES‐buffered saline (sterile; see recipe)
  • Growth medium (see recipe)
  • 15‐ml round‐bottom polystyrene tubes (sterile)
  • Pasteur pipets (autoclaved)
  • Incubators, humidified 37°C, 3% and 5% CO 2

Support Protocol 1: Culturing Rat Cerebellar Granule Neurons

  Materials
  • Silanizing solution (e.g., Sigmacote, Sigma)
  • 0.1 to 1 mg/ml poly‐D‐lysine (mol. wt. >150,000 kDa) in sterile water
  • 4‐ to 10‐day‐old rat pups
  • 70% (v/v) ethanol
  • Ca2+, Mg2+‐free HBSS/pen/strep, pH 7.4 (see recipe), ice‐cold
  • 0.5 g/liter trypsin solution (see recipe)
  • Fetal bovine serum (FBS; appendix 2A)
  • Growth medium (see recipe)
  • Tissue culture plates (35‐, 60‐, or 100‐mm, depending on the final use) or glass coverslips in 35‐mm plates
  • Dissecting tools (fine forceps, large forceps, fine dissecting scissors, iris scissors, large dissecting scissors), autoclaved
  • 60‐mm petri plates, sterile
  • #11 and #15 scalpels, sterile
  • 15‐ml polystyrene tubes, sterile
  • Incubator, humidified 37°C, 5% CO 2
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Arbert‐Engels, C., Tartare‐Deckert, S., and Eckhart, W. 1999. C‐terminal Src kinase associates with ligand‐stimulated insulin‐like growth factor‐1 receptor. J. Biol. Chem. 274:5422‐5428.
   Blair, L.A.C. and Marshall, J. 1997. IGF‐1 modulates N and L calcium channels in a PI 3‐kinase‐dependent manner. Neuron 19:421‐429.
   Blair, L.A.C., Bence‐Hanulec, , K.K., Mehta, S., Franke, T., Kaplan, D., and Marshall, J. 1999a. Akt‐dependent potentiation of L channels by IGF‐1 is required for neuronal survival. J. Neurosci. 19:1940‐1951.
   Blair, L.A.C., Bence‐Hanulec, K.K., and Marshall, J. 1999b. The jellyfish green fluorescent protein: A tool for studying ion channels and second messenger signalling in neurons. Methods Enzymol. 302:213‐225.
   Chen, C. and Okayama, H. 1987. High efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7:2745‐2752.
   Cohick, W.S. and Clemmons, D.R. 1993. The insulin‐like growth factors. Annu. Rev. Physiol. 55:131‐153.
   Cubitt, A.B., Heim, R., Adams, S.R., Boyd, A.E., Gross, L.A., and Tsien, R.Y. 1995. Understanding, improving and using green fluorescent proteins. Trends Biochem. Sci. 20:448‐455.
   Delbono, O., Renganathan, M., and Messi, M.L. 1997. Regulation of mouse skeletal muscle L‐type Ca2+ channel by activation of the insulin‐like growth factor‐1 receptor. J. Neurosci. 17:6918‐6928.
   Dudek, H., Datta, S.R., Franke, T.F., Birnbaum, M.J., Yao, R., Cooper, G.M., Segal, R.A., Kaplan, D.R., and Greenberg, M.E. 1997. Regulation of neuronal survival by the serine‐threonine protein kinase Akt. Science 275:661‐665.
   Finkbeiner, S. and Greenberg, M.E. 1996. Ca+2‐dependent routes to Ras: Mechanisms for neuronal survival, differentiation, and plasticity? Neuron 16:233‐236.
   Franke, T.F., Yang, S.‐I., Chan, T.O., Datta, K., Kazlauskas, A., Morrison, D.K., Kaplan, D.R., and Tsichlis, P.N. 1995. The protein kinase encoded by the Akt proto‐oncogene is a target of PDGF‐activated phosphatidylinositol 3‐kinase. Cell 81:727‐736.
   Goslin, K. and Banker, G. 1991. Rat hippocampal neurons in low density. In Culturing Nerve Cells (G. Banker and K. Goslin, eds.) pp. 251‐282. MIT Press, Cambridge, Mass.
   Heim, R. and Tsien, R. 1996. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr. Biol. 6:178‐182.
   Hilborn, M.D., Vaillancourt, R.R., and Rane, S.G. 1998. Growth factor receptor tyrosine kinases acutely regulate neuronal sodium channels through the src signaling pathway. J. Neurosci. 18:590‐600.
   Kim, B., Cheng, H.L., Margolis, B., and Feldman, E.L. 1998. Insulin receptor substrate 2 and Shc play different roles in insulin‐like growth factor I signaling. J. Biol. Chem. 273:34543‐34550.
   Klint, P., Kanda, S., Koog, Y., and Claesson‐Welsh, L. 1999. Contribution of Src and Ras pathways in FGF‐2 induced endothelial cell differentiation. Oncogene 18:3354‐3364.
   Marshall, J., Molloy, R., Moss, G.W.J., Howe, J.R., and Hughes, T.E. 1995. The jellyfish green fluorescent protein: A new tool for studying ion channel expression and function. Neuron 14:211‐215.
   Messer, A. 1972. The maintenance and identification of mouse cerebellar granule cells in monolayer culture. Brain Res. 130:1‐12.
   Palm, G.J., Zdanov, A., Gaitanaris, G.A., Stauber, R., Pavlakis, G.N., and Wlodawer, A. 1997. The structural basis for spectral variations in green fluorescent protein. Nature Struct. Biol. 4:361‐365.
   Prasher, D.C., Eckenrode, V.K., Ward, W.W., Prendergast, F.G., and Cormier, M.J. 1992. Primary structure of the Aequorea victoria green‐fluorescent protein. Gene 111:229‐233.
   Qu, C.K., Yu, W.M., Azzarelli, B., and Feng, G.S. 1999. Genetic evidence that Shp‐2 tyrosine phosphatase is a signal enhancer of the epidermal growth factor receptor in mammals. Proc. Natl. Acad. Sci. U.S.A. 96:8528‐8533.
   Stauber, R.H., Horie, K., Carney, P., Hudson, E.A., Tarasova, N.I., Gaitanaris, G.A., and Pavlakis, G.N. 1998. Development and applications of enhanced green fluorescent protein mutants. BioTechniques 24:462‐472.
   Wachter, R.M., King, B.A., Heim, R., Kallio, K., Tsien, R.Y., Boxer, S.G., and Remington, S.J. 1997. Crystal structure and photodynamic behavior of the blue emission variant Y66H/Y145F of green fluorescent protein. Biochemistry 36:9759‐9765.
   Wang, J., Dai, H., Yousaf, N., Moussaif, M., Deng, Y., Boufelliga, A., Swamy, O.R., Leone, M.E., and Riedel, H. 1999. Grb‐10, a positive, stimulatory signaling adapter in platelet‐derived growth factor BB‐, insulin‐like growth factor I‐, and insulin‐mediated mitogenesis. Mol. Cell. Biol. 19:6217‐6228.
   Wu, H., Reuver, S.M., Kuhlendahl, S., Chung, W.J., and Garner, C.C. 1998. Subcellular targeting and cytoskeletal attachment of SAP97 to the epithelial lateral membrane. J. Cell Sci. 111:2365‐2376.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library