Gene discovery in genetically labeled single dopaminergic neurons of the retina

Stefano Gustincich, Massimo Contini, Manuela Gariboldi, Michelino Puopolo, Koji Kadota, Hidemasa Bono, Julianna LeMieux, Pamela Walsh, Piero Carninci, Yoshihide Hayashizaki, Yasushi Okazaki, Elio Raviola

Research output: Contribution to journalArticle

Abstract

In the retina, dopamine plays a central role in neural adaptation to light. Progress in the study of dopaminergic amacrine (DA) cells has been limited because they are very few (450 in each mouse retina, 0.005% of retinal neurons). Here, we applied transgenic technology, single-cell global mRNA amplification, and cDNA microarray screening to identify transcripts present in DA cells. To profile gene expression in single neurons, we developed a method (SMART7) that combines a PCR-based initital step (switching mechanism at the 5′ end of the RNA transcript or SMART) with T7 RNA polymerase amplification. Single-cell targets were synthesized from genetically labeled DA cells to screen the RIKEN 19k mouse cDNA microarrays. Seven hundred ninety-five transcripts were identified in DA cells at a high level of confidence, and expression of the most interesting genes was confirmed by immunocytochemistry. Twenty-one previously undescribed proteins were found in DA cells, including a chloride channel, receptors and other membrane glycoproteins, kinases, transcription factors, and secreted neuroactive molecules. Thirty-eight percent of transcripts were ESTs or coding for hypothetical proteins, suggesting that a large portion of the DA cell proteome is still uncharacterized. Because cryptochrome-1 mRNA was found in DA cells, immunocytochemistry was extended to other components of the circadian clock machinery. This analysis showed that DA cells contain the most common clock-related proteins.

Original languageEnglish
Pages (from-to)5069-5074
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume101
Issue number14
DOIs
Publication statusPublished - Apr 6 2004

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ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Gustincich, S., Contini, M., Gariboldi, M., Puopolo, M., Kadota, K., Bono, H., LeMieux, J., Walsh, P., Carninci, P., Hayashizaki, Y., Okazaki, Y., & Raviola, E. (2004). Gene discovery in genetically labeled single dopaminergic neurons of the retina. Proceedings of the National Academy of Sciences of the United States of America, 101(14), 5069-5074. https://doi.org/10.1073/pnas.0400913101