Selective blockade of gene expression in a single identified snail neuron.
In the present study, the applicability of antisense morpholino oligos for loss-of-function experiments in neurobiology was investigated. The identified withdrawal interneurons of the parietal ganglia expressing helix command neuron-specific 2 (HCS2) gene were pressure injected with HCS2 antisense or control morpholino oligo solution at a final concentration 1-4 microM. No toxic or side effects for the neural functioning were noted immediately or several hours after injection. The changes in the concentration of HCS2-encoded protein in neurons after injection were monitored by two methods, Western blotting and immunostaining of the brain. The amount of the peptide immunoreactive with the HCS2 antibody started to decline in the injected cells at day 2 post-injection, decreased four- to five-fold at day 4, and stayed at this low level thereafter. Similar results obtained by both methods suggest significant selective blockade of production of the HCS2-encoded peptide. In contrast, no substantial decrease of the HCS2-encoded polypeptide was observed after injection with control oligos. Due to the high stability of the morpholino oligos in the cell, they represent a highly efficient tool for a specific long-term blockade of gene expression in molluscan neurons.
Boguslavsky
D
D
Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, Butlerova 5A, Moscow 117865, Russia.
Ierusalimsky
V
V
Malyshev
A
A
Balaban
P
P
Belyavsky
A
A
D000818:Animals; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology; D004305:Dose-Response Relationship, Drug; D017952:Ganglia, Invertebrate / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D005786:Gene Expression Regulation / Q000187:drug effects*; D006372:Helix, Snails; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D009025:Morpholines / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects* / Q000502:physiology; D009479:Neuropeptides / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology*; D010446:Peptide Fragments / Q000276:immunology; D013997:Time Factors
D000906:Antibodies; D002135:Calcium-Binding Proteins; C116258:HCS2 protein, Helix lucorum; D009025:Morpholines; D009479:Neuropeptides; D020319:Oligodeoxyribonucleotides, Antisense; D010446:Peptide Fragments