title: The production of viral vectors designed to express large and difficult to express transgenes within neurons.
abstract: BACKGROUND
  Viral vectors are frequently used to deliver and direct expression of transgenes in a spatially and temporally restricted manner within the nervous system of numerous model organisms. Despite the common use of viral vectors to direct ectopic expression of transgenes within the nervous system, creating high titer viral vectors that are capable of expressing very large transgenes or difficult to express transgenes imposes unique challenges. Here we describe the development of adeno-associated viruses (AAV) and lentiviruses designed to express the large and difficult to express GluN2A or GluN2B subunits of the N-methyl-D-aspartate receptor (NMDA) receptor, specifically within neurons.
  
  
  RESULTS
  We created a number of custom designed AAV and lentiviral vectors that were optimized for large transgenes, by minimizing DNA sequences that were not essential, utilizing short promoter sequences of 8 widely used promoters (RSV, EFS, TRE3G, 0.4αCaMKII, 1.3αCaMKII, 0.5Synapsin, 1.1Synapsin and CMV) and utilizing a very short (~75 bps) 3' untranslated sequence. Not surprisingly these promoters differed in their ability to express the GluN2 subunits, however surprisingly we found that the neuron specific synapsin and αCaMKII, promoters were incapable of conferring detectable expression of full length GluN2 subunits and detectable expression could only be achieved from these promoters if the transgene included an intron or if the GluN2 subunit transgenes were truncated to only include the coding regions of the GluN2 transmembrane domains.
  
  
  CONCLUSIONS
  We determined that viral packaging limit, transgene promoter and the presence of an intron within the transgene were all important factors that contributed to being able to successfully develop viral vectors designed to deliver and express GluN2 transgenes in a neuron specific manner. Because these vectors have been optimized to accommodate large open reading frames and in some cases contain an intron to facilitate expression of difficult to express transgenes, these viral vectors likely could be useful for delivering and expressing many large or difficult to express transgenes in a neuron specific manner.
authors.affiliation: School of Behavioral and Brain Sciences and the Department of Molecular & Cell Biology, University of Texas at Dallas, 800 West Campbell Road, Richardson, TX, 75080, USA. roopa.hs@gmail.com.
authors.forename: Roopashri
authors.initials: R
authors.lastname: Holehonnur
authors.affiliation: School of Behavioral and Brain Sciences and the Department of Molecular & Cell Biology, University of Texas at Dallas, 800 West Campbell Road, Richardson, TX, 75080, USA. sklella@yahoo.com.
authors.forename: Srihari K
authors.initials: SK
authors.lastname: Lella
authors.affiliation: School of Behavioral and Brain Sciences and the Department of Molecular & Cell Biology, University of Texas at Dallas, 800 West Campbell Road, Richardson, TX, 75080, USA. axh097020@utdallas.edu.
authors.forename: Anthony
authors.initials: A
authors.lastname: Ho
authors.affiliation: School of Behavioral and Brain Sciences and the Department of Molecular & Cell Biology, University of Texas at Dallas, 800 West Campbell Road, Richardson, TX, 75080, USA. jojo2049@gmail.com.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Luong
authors.affiliation: School of Behavioral and Brain Sciences and the Department of Molecular & Cell Biology, University of Texas at Dallas, 800 West Campbell Road, Richardson, TX, 75080, USA. Jonathan.Ploski@UTDallas.edu.
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Ploski
mesh_terms: D000818:Animals; D000229:Dependovirus / Q000378:metabolism; D005822:Genetic Vectors / Q000378:metabolism*; D016679:Genome, Viral; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007438:Introns / Q000235:genetics; D016086:Lentivirus / Q000378:metabolism*; D008297:Male; D008810:Mice, Inbred C57BL; D050505:Mutant Proteins / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D052736:Rous sarcoma virus / Q000378:metabolism; D019076:Transgenes
chemical_list: D050505:Mutant Proteins; D016194:Receptors, N-Methyl-D-Aspartate; D049452:Green Fluorescent Proteins