In vivo somatic delivery of plasmid DNA and retrograde transport to obtain cell-specific gene expression in the central nervous system.
We utilised the retrograde transport machinery of neurones to deliver naked plasmid DNA into the central nervous system. A 5.4-kb fragment of the glycine receptor (GlyR) alpha1 subunit gene was cloned and used to drive the expression of a construct encoding for the enhanced green fluorescent protein (EGFP). Injections of the plasmid DNA in the tongue of mice resulted in the expression of the marker protein in hypoglossal motor neurones, showing that the GlyRalpha1 promoter sequence is sufficient to drive expression of the transgene. In order to determine the specificity of expression of the 5.4-kb fragment of the GlyR alpha1 subunit gene promoter, we subsequently injected the plasmid DNA into the mouse central nucleus of the amygdala. This nucleus receives projections from the parabrachial nucleus, a brainstem area that has a high density of GlyRs, and from the insular cortex, a forebrain structure devoid of GlyRs. We observed EGFP-labelled neurones in the parabrachial nucleus, but not in the insular cortex, indicating that the 5.4-kb GlyR alpha1 subunit gene promoter confers specificity of expression. This approach provides a simple and rapid way to identify, in vivo, promoter elements that mediate neurone-specific gene expression.
Morris
Renée
R
Garvan Institute of Medical Research, Sydney, New South Wales, Australia.
Morgan
Branwen S
BS
Lewis
Trevor M
TM
Pierce
Kerrie D
KD
Pisano
Anthony
A
Schofield
Peter R
PR
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002460:Cell Line; D002490:Central Nervous System / Q000166:cytology* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000378:metabolism*; D004622:Embryo, Mammalian; D015870:Gene Expression / Q000502:physiology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006651:Histocytochemistry / Q000379:methods; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000378:metabolism*; D010751:Phosphopyruvate Hydratase / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000502:physiology; D018009:Receptors, Glycine / Q000235:genetics / Q000378:metabolism*; D014059:Tongue / Q000294:innervation / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology*
D008164:Luminescent Proteins; D018009:Receptors, Glycine; D049452:Green Fluorescent Proteins; D004247:DNA; D010751:Phosphopyruvate Hydratase