Exogenous mRNA encoding tetanus or botulinum neurotoxins expressed in Aplysia neurons.

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RESUMO

Injection of exogenous mRNA purified from various tissue preparations into cellular translation systems such as Xenopus oocytes has allowed expression of complex proteins (e.g., receptors for neurotransmitters). No evidence for expression of injected exogenous mRNA, however, has been reported in terminally differentiated neurons. If achieved, it would allow the study of long-lasting changes of properties of nerve cells in their functional context. To obtain evidence of such expression, we chose two proteins that produce a detectable effect even at very low intracellular concentrations. Tetanus toxin and botulinum neurotoxin fulfill this criterion, being the most potent neurotoxins known. Both toxins block neurotransmitter release at nanomolar intracellular concentrations. These di-chain proteins, consisting of a light chain and a heavy chain, have recently been sequenced. Their active sites are located (or partly located) on the light chain. mRNAs encoding the light chain of either toxin were transcribed in vitro from the cloned and specifically truncated genes of Clostridium tetani and Clostridium botulinum, respectively, and injected into presynaptic cholinergic neurons of the buccal ganglia of Aplysia californica. Depression of neurotransmitter release appeared in less than 1 hr, demonstrating successful expression of foreign mRNA injected into a neuron in situ.

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