The Biological Bulletin, Vol 197, Issue 1 40-48, Copyright © 1999 by Marine Biological Laboratory

NEUROBIOLOGY AND BEHAVIOR

Long-Term Culture of Lobster Central Ganglia: Expression of Foreign Genes in Identified Neurons

G. K. Ganter, R. Heinrich, R. P. Bunge and E. A. Kravitz
Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, Massachusetts 02115

The ventral nerve cords of lobsters (Homarus americanus) can be cultured in vitro for at least 7 weeks. Over this period, neurons maintain their normal electrophysiological features and continue, among other measures of neuronal health, to synthesize RNA and proteins. One application of this culture system is demonstrated: the manipulation of gene expression in identified neurons. After intracellular injection of complementary RNA (cRNA) encoding green fluorescent protein (GFP), the amount of protein product measured by fluorescent confocal microscopy increases for 4 days and then decreases to background by day 10. Thus, translation of the injected message must have increased for 4 days before declining. Moreover, after injection of cRNA encoding {beta}-galactosidase, the levels of enzyme activity were measured using a fluorogenic substrate, revealing a peak of {beta}-galactosidase activity at 6 to 9 days; this activity was still detectable for at least 10 days after injection. Therefore, either GFP or {beta}-galactosidase can be used as an injectable marker, allowing in vivo quantitation of expression in individual cells over time. We measured long-lasting expression of these proteins after a single injection, suggesting that it may be possible to manipulate the levels of expression of any gene of interest.

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