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.
|Titolo:||Gene discovery in genetically labeled single dopaminergic neurons of the retina|
|Autori:||Gustincich, Stefano; Contini, M.; Gariboldi, M.; Puopolo, M.; Kadota, K.; Bono, H.; Lemieux, J.; Walsh, P.; Carninci, P.; Hayashizaki, Y.; Okazaki, Y.; Raviola, E.|
|Data di pubblicazione:||2004|
|Digital Object Identifier (DOI):||10.1073/pnas.0400913101|
|Appare nelle tipologie:||1.1 Journal article|
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