title: pUNISHER: a high-level expression cassette for use with recombinant viral vectors for rapid and long term in vivo neuronal expression in the CNS.
abstract: Fast onset and high-level neurospecific transgene expression in vivo is of importance for many areas in neuroscience, from basic to translational, and can significantly reduce the amount of vector load required to maintain transgene expression in vivo. In this study, we tested various cis elements to optimize transgene expression at transcriptional, posttranscriptional, and posttranslational levels and combined them together to create the high-level neuronal transgene expression cassette pUNISHER. Using a second-generation adenoviral vector system in combination with the pUNISHER cassette, we characterized its rate of onset of detectable expression and levels of expression compared with a neurospecific expression cassette driven by the 470-bp human synapsin promoter in vitro and in vivo. Our results demonstrate in primary neurons that the pUNISHER cassette, in a recombinant adenovirus type 5 background, led to a faster rate of onset of detectable transgene expression and higher level of transgene expression. More importantly, this cassette led to highly correlated neuronal expression in vivo and to stable transgene expression up to 30 days in the auditory brain stem with no toxicity on the characteristics of synaptic transmission and plasticity at the calyx of Held synapse. Thus the pUNISHER cassette is an ideal high-level neuronal expression cassette for use in vivo for neuroscience applications.
authors.affiliation: Research Group Molecular Mechanisms of Synaptic Function, Max Planck Florida Institute, 5353 Parkside Drive MC19-RE, Jupiter, FL 33458, USA.
authors.forename: Monica S
authors.initials: MS
authors.lastname: Montesinos
authors.forename: Zuxin
authors.initials: Z
authors.lastname: Chen
authors.forename: Samuel M
authors.initials: SM
authors.lastname: Young
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000378:metabolism; D002461:Cell Line, Transformed; D002490:Central Nervous System / Q000166:cytology*; D003587:Cytomegalovirus / Q000235:genetics; D019706:Excitatory Postsynaptic Potentials / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D018014:Gene Transfer Techniques / Q000295:instrumentation; D005822:Genetic Vectors / Q000008:administration & dosage*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000235:genetics; D051379:Mice; D009474:Neurons / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010751:Phosphopyruvate Hydratase / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012323:RNA Processing, Post-Transcriptional; D051381:Rats; D017208:Rats, Wistar; D012045:Regulatory Sequences, Nucleic Acid; D051670:Shaw Potassium Channels / Q000235:genetics / Q000378:metabolism; D013569:Synapses / Q000235:genetics / Q000378:metabolism; D016704:Synapsins / Q000235:genetics / Q000378:metabolism; D009435:Synaptic Transmission / Q000235:genetics; D013997:Time Factors; D014162:Transfection
chemical_list: D051670:Shaw Potassium Channels; D016704:Synapsins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D010751:Phosphopyruvate Hydratase