[讨论]Gene discovery in genetica

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 and Elio Raviola * ||

*Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115; Genome Exploration Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan; and ¶RIKEN Genomic Sciences Laboratory, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

Communicated by David H. Hubel, Harvard Medical School, Boston, MA, February 9, 2004 (received for review December 17, 2003)


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.