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Analysis of Gene Expression in Genetically Labeled Single Cells

  • Stefano Gustincich
  • Andreas Feigenspan
  • Elio Raviola

Abstract

A combination of transgenic technology and single-cell reverse transcription polymerase chain reaction (RT-PCR)has been used to study gene expression in dopaminergic amacrine (DA)cells of the mouse retina. Because there are only 900 DA cells, and they cannot be distinguished from neighboring neurons on the basis of their morphology, we labeled them with human placental alkaline phosphatase (PLAP)by introducing into the mouse genome PLAP cDNA under the control of the promoter of the gene for tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine biosynthesis. Because PLAP is an enzyme that resides on the outer surface of the cell membrane, we can identify DA cells after dissociation of the retina by immunocytochemistry in the living state. Cells are then patch clamped and harvested for single-cell RT-PCR analysis of gene expression. Here, we describe the preparation of the fluorescent antibody E6-Cy3 to specifically detect PLAP-expressing cells, methods to obtain short-term cultures of solitary neurons from mouse retinas, and techniques to detect gene expression in individual neurons. Properties and pitfalls of single-cell RT-PCR are described and discussed.

Keywords

Tyrosine Hydroxylase Amacrine Cell Inner Nuclear Layer Mouse Retina Tyrosine Hydroxylase 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.

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References

  1. 1.
    Audinat, E., B. Lambolez, and J. Rossier. 1996. Functional and molecular analysis of glutamate-gated channels by patch-clamp and RT-PCR at the single cell level. Neurochem. Int. 28:119–136.PubMedCrossRefGoogle Scholar
  2. 2.
    Banerjee, S.A., P. Hoppe, M. Brilliant, and D.M. Chikaraishi. 1992. 5’ Flanking sequences of the rat tyrosine hydroxylase gene target accurate tissue-specific, developmental, and transsynaptic expression in transgenic mice. J. Neurosci. 12:4460–4467.PubMedGoogle Scholar
  3. 3.
    Berger, J., A.D. Howard, L. Gerber, B.R. Cullen, and S. Udenfriend. 1987. Expression of active, membrane-bound human placental alkaline phosphatase by transfected simian cells. Proc. Natl. Acad. Sci. USA 84:4885–4889.PubMedCrossRefGoogle Scholar
  4. 4.
    Bochet, P., E. Audinat, B. Lambolez, F. Crepel, J. Rossier, M. Iino, K. Tsuzuki, and S. Ozawa. 1994. Subunit composition at the single-cell level explains functional properties of a glutamate-gated channel. Neuron 12:383–388.PubMedCrossRefGoogle Scholar
  5. 5.
    Chalfie, M., Y. Tu, G. Euskirchen, W.W. Ward, and D.C. Prasher. 1994. Green fluorescent protein as a marker for gene expression. Science 263:802–805.PubMedCrossRefGoogle Scholar
  6. 6.
    Estee, J., P. Crino, and J. Eberwine. 1999. Preparation of cDNA from single cells and subcellular regions, p. 3–18. In S.M. Weissman (Ed.), Methods of Enzymology (303): cDNA Preparation and Characterization. Academic Press, San Diego.Google Scholar
  7. 7.
    De Groote G., P. De Waele, A. Van De Voorde, M. De Broe, and W. Fiers. 1983. Use of monoclonal antibodies to detect human placental alkaline phosphatase. Clin. Chem. 29:115–119.PubMedGoogle Scholar
  8. 8.
    DeVries, S.H. and D.A. Baylor. 1997. Mosaic arrangement of ganglion cell receptive fields in rabbit retina. J. Neurophysiol. 78:2048–2060.PubMedGoogle Scholar
  9. 9.
    De Waele, P., G. De Groote, A. Van De Voorde, W Fiers, J.-D. Franssen, P. Herion, and J. Urbain. 1982. Isolation and identification of monoclonal antibodies directed against human placental alkaline phosphatase. Arch. Int. Physiol. Biochim. 90:B21.CrossRefGoogle Scholar
  10. l0.
    Djamgoz, M.B., M.W Hankins, J. Hirano, and S.N. Archer. 1997. Neurobiology of retinal dopamine in relation to degenerative states of the tissue. Vision Res. 37:3509–3529.PubMedCrossRefGoogle Scholar
  11. ll.
    Euler, T., H. Schneider, and H. Wassle. 1996. Glutamate responses of bipolar cells in a slice preparation of the rat retina. J. Neurosci. 16:2934–2944.PubMedGoogle Scholar
  12. 12.
    Feigenspan, A., S. Gustincich, B.P. Bean, and E. Raviola. 1998. Spontaneous activity of solitary dopaminergic cells of the retina. J. Neurosci. 18:6776–6789.PubMedGoogle Scholar
  13. 13.
    Fields-Berry, S.C., A.L. Halliday, and C.L. Cepko. 1992. A recombinant retrovirus encoding alkaline phosphatase confirms clonal boundary assignment in lineage analysis of murine retina. Proc. Natl. Acad. Sci. USA 89:693–697.PubMedCrossRefGoogle Scholar
  14. 14.
    Geiger, J.R., T. Melcher, D.S. Koh, B. Sakmann, P.H. Seeburg, P. Jonas, and H. Monyer. 1995. Relative abundance of subunit mRNAs determines gating and Ca2+ permeability of AMPA receptors in principal neurons and interneurons in rat CNS. Neuron 15:193–204.PubMedCrossRefGoogle Scholar
  15. 15.
    Gustincich, S., D.K. Wu, L.J. Koopman, and E. Raviola 1996. A transgenic approach to the study of neural al networks in the retina. Invest. Ophthalmol. Vis. Sci. 37:S1060.Google Scholar
  16. 16.
    Gustincich, S., A. Feigenspan, D.K. Wu, L.J. Koopman, and E. Raviola. 1997. Control of dopamine release in the retina: a transgenic approach to neural networks. Neuron 18:723–736.PubMedCrossRefGoogle Scholar
  17. 17.
    Gustincich, S., A. Feigenspan, W Sieghart, and E. Raviola. 1999. Composition of the GABA(A) receptors of retinal dopaminergic neurons. J. Neurosci. 19:7812–7822.PubMedGoogle Scholar
  18. 18.
    Harlow, E. and D. Lane. 1988. Antibodies: A Laboratory Manual. CSH Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  19. 19.
    Jonas P., C. Racca, B. Sakmann, P.H. Seeburg, and H. Monyer. 1994. Differences in Ca2+ permeability of AMPA-type glutamate receptor channels in neocortical neurons caused by differential GluR-B subunit expression. Neuron 12:1281–1289.PubMedCrossRefGoogle Scholar
  20. 20.
    Kam, W., E. Clauser, Y.S. Kim, Y.W. Kan, and W.J. Rutter. 1985. Cloning, sequencing, and chromosomal localization of human term placental alkaline phosphatase cDNA. Proc. Natl. Acad. Sci. USA 82:8715–8719.PubMedCrossRefGoogle Scholar
  21. 21.
    Lam, D.M. 1972. Biosynthesis of acetylcholine in turtle photoreceptors. Proc. Natl. Acad. Sci. USA 69: 1987–1991.PubMedCrossRefGoogle Scholar
  22. 22.
    Lambolez, B., E. Audinat, P. Bochet, F. Crepel, and J. Rossier. 1992. AMPA receptor sub units expressed by single Purkinje cells. Neuron 9:247–258.PubMedCrossRefGoogle Scholar
  23. 23.
    MacGregor, G.R., A.E. Mogg, J.F. Burke, and C.T. Caskey. 1987. Histochemical staining of clonal mammalian cell lines expressing E. coli beta-galactosidase indicates heterogeneous expression of the bacterial gene. Somat. Cell Mol. Genet. 13:253–65.PubMedCrossRefGoogle Scholar
  24. 24.
    MacNeil, M.A., J.K Heussy, R.F. Dacheux, E. Raviola, and R.H. Masland. 1999. The shapes and numbers of amacrine cells: matching of photofilled with golgistained cells in the rabbit retina and comparison with other mammalian species. J. Comp. Neurol. 413:305–326.PubMedCrossRefGoogle Scholar
  25. 25.
    Masland, R.H. and E. Raviola. 2000. Confronting complexity: strategies for understanding the microcircuitry of the retina. Ann. Rev. Neurosci. 23:249–284.PubMedCrossRefGoogle Scholar
  26. 26.
    Mayahara, H., H. Hirano, T. Saito, and K Ogawa. 1967. The new lead citrate method for the ultracytochemical demonstration of activity of non-specific alkaline phosphatase (orthophosphoric monoester phosphohydrolase). Histochemie 11:88–96.PubMedCrossRefGoogle Scholar
  27. 27.
    McComb, R.B. and G.N. Bowers, Jr. 1972. Study of optimum buffer conditions for measuring alkaline phosphatase activity in human serum. Clin. Chem. 18:97–104.PubMedGoogle Scholar
  28. 28.
    Monyer, H. and B. Lambolez. 1995. Molecular biology and physiology at the single-cell level. Curr. Opin. Neurobiol. 5:382–387.PubMedCrossRefGoogle Scholar
  29. 29.
    Oberdick, J., R.J. Smeyne, J.R. Mann, S. Zackson, and J.I. Morgan. 1990. A promoter that drives transgene expression in cerebellar Purkinje and retinal bipolar neurons. Science 248:223–226.PubMedCrossRefGoogle Scholar
  30. 30.
    O’Dowd, D.K. and M.A. Smith. 1996. Single-cell analysis of gene expression in the nervous system. Measurements at the edge of chaos. Mol. Neurobiol. 13:199–211.PubMedCrossRefGoogle Scholar
  31. 31.
    Parra, P., A.I. Gulyas, and R. Miles. 1998. How many subtypes of inhibitory cells in the hippocampus? Neuron 20:983–993.PubMedCrossRefGoogle Scholar
  32. 32.
    Posen, S., C.J. Cornish, M. Home, and P.K. Saini. 1969. Placental alkaline phosphatase and pregnancy. Ann. NY Acad. Sci. 166:733–774.PubMedCrossRefGoogle Scholar
  33. 33.
    Spergel, D.J., U. Kruth, D.E. Hanley, R. Sprengel, and P.H. Seeburg. 1999. GABA-and glutamate-activated channels in green fluorescent protein-tagged gonadotropin-releasing hormone neurons in transgenic mice. J. Neurosci. 19:2037–2050.PubMedGoogle Scholar
  34. 34.
    Stevens, C.F. 1998. Neuronal diversity: too many cell types for comfort? Curr. Biol. 8:R708–R710.PubMedCrossRefGoogle Scholar
  35. 35.
    Sucher, N.J. and D.L. Deitcher. 1995. PCR and patch-clamp analysis of single neurons. Neuron 14:1095–1100.PubMedCrossRefGoogle Scholar
  36. 36.
    Tsien, R.Y. 1998. The green fluorescent protein. Ann. Rev. Biochem. 67:509–544.PubMedCrossRefGoogle Scholar
  37. 37.
    Van Gelder, R.N., M.E. von Zastrow, A. Yool, W.C. Dement, J.D. Barchas, and J.H. Eberwine. 1990. Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc. Natl. Acad. Sci. USA 87:1663–1667.PubMedCrossRefGoogle Scholar
  38. 38.
    Versaux-Botteri, C.,J. Nguyen-Legros, A. Vigny, and N. Raoux. 1984. Morphology, density and distribution of tyrosine hydroxylase-like immunoreactive cells in the retina of mice. Brain Res. 301:192–197.PubMedCrossRefGoogle Scholar
  39. 39.
    Witkovsky, P. and A. Dearry. 1991. Functional roles of dopamine in the vertebrate retina. Progr. Retinal Res. 11:247–292.CrossRefGoogle Scholar
  40. 40.
    Zlokarnik, G., P.A. Negulescu, T.E. Knapp, L. Mere, N. Burres, L. Feng, M. Whitney, K. Roemer, and R.Y. Tsien. 1998. Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter. Science 279:84–88.PubMedCrossRefGoogle Scholar
  41. 41.
    Zolotukhin, S., M. Potter, W.W. Hauswirth, J. Guy, and N. Muzyczka. 1996. A “humanized” green fluorescent protein cDNA adapted for high-level expression in mammalian cells. J. Virol. 70:4646–4654.PubMedGoogle Scholar

Copyright information

© Springer-Verlag New York, Inc. 2002

Authors and Affiliations

  • Stefano Gustincich
    • 1
  • Andreas Feigenspan
    • 1
  • Elio Raviola
    • 1
  1. 1.Department of NeurobiologyHarvard Medical SchoolBostonUSA

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