Adenoviral-mediated, high-level, cell-specific transgene expression: a SYN1-WPRE cassette mediates increased transgene expression with no loss of neuron specificity.
Viral vectors are excellent tools for studying gene function in the brain, although a limitation has been the ability to effectively target transgene expression to specific neuronal populations. This generally cannot be overcome by the use of neuron-specific promoters, as most are too large to be used with current viral vectors and expression from these promoters is often relatively weak. We therefore developed a composite expression cassette, comprising 495 bp of the weak human SYN1 (synapsin-1) promoter and 800 bp of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Studies in hippocampal cultures, organotypic cultures, and in vivo showed that the 3' addition of the WPRE to the SYN1 element greatly increased enhanced green fluorescent protein expression levels with no loss of neuronal specificity. In vivo studies also showed that transgene expression was enhanced with no loss of neuronal specificity in dentate-gyrus neurons for at least 6 weeks following transfection. Therefore, unlike most powerful promoter systems, which mediate expression in neurons and glia, this SYN1-WPRE cassette can target powerful long-term transgene expression to central nervous system neurons when delivered at relatively low titers of adenovirus. Its use should therefore facilitate both gene therapy studies and investigations of neuronal gene function.
Glover
Colin P J
CP
MRC Centre for Synaptic Plasticity and University Research Centre for Neuroendocrinology, University of Bristol, Bristol, BS2 8HW, UK.
Bienemann
Alison S
AS
Heywood
Darren J
DJ
Cosgrave
A Siobhan
AS
Uney
James B
JB
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D005434:Flow Cytometry; D015967:Gene Expression Regulation, Viral; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000008:administration & dosage* / Q000235:genetics; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D017919:Hepatitis B Virus, Woodchuck / Q000235:genetics*; D006624:Hippocampus / Q000378:metabolism*; D006801:Humans; D007124:Immunoenzyme Techniques; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008297:Male; D009474:Neurons / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012323:RNA Processing, Post-Transcriptional; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D012045:Regulatory Sequences, Nucleic Acid; D016704:Synapsins / Q000235:genetics* / Q000378:metabolism*; D019076:Transgenes
D008164:Luminescent Proteins; D012367:RNA, Viral; D016704:Synapsins; D049452:Green Fluorescent Proteins