Assessment of CMV, RSV and SYN1 promoters and the woodchuck post-transcriptional regulatory element in adenovirus vectors for transgene expression in cortical neuronal cultures.
In order to investigate protein function in rat primary cortical neuronal cultures, we modified an adenoviral vector expression system and assessed the strength and specificity of the cytomegalovirus (CMV), rous sarcoma virus (RSV), and rat and human synapsin 1 (SYN1) promoters to drive DsRed-X expression. We also incorporated the woodchuck post-transcriptional regulatory element (WPRE) and a CMV promoter-enhanced green fluorescent protein (EGFP) reporter cassette. We observed that the RSV promoter activity was strong in neurons and moderate in astrocytes, while the CMV promoter activity was weak-to-moderate in neurons and very strong in astrocytes. The rat and human SYN1 promoters exhibited similar but weak activity in neurons, despite inclusion of the WPRE. We confirmed that the WPRE enhanced RSV promoter-mediated DsRed-X expression in a time-dependent fashion. Interestingly, we observed very weak SYN1-mediated DsRed-X expression in astrocytes and HEK293 cells suggesting incomplete neuronal-restrictive behavior for this promoter. Finally, using our adenoviral expression system, we demonstrated that RSV promoter-mediated Bcl-X(L) overexpression attenuated neuronal death caused by in vitro ischemia and oxidative stress.
Boulos
Sherif
S
Centre for Neuromuscular and Neurological Disorders, The University of Western Australia, and Australian Neuromuscular Research Institute. sboulous@cyllene.uwa.edu.au
Meloni
Bruno P
BP
Arthur
Peter G
PG
Bojarski
Christina
C
Knuckey
Neville W
NW
D000818:Animals; D001253:Astrocytes / Q000378:metabolism / Q000821:virology; D001358:Avian Sarcoma Viruses / Q000235:genetics*; D001555:Benzene Derivatives / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D016923:Cell Death / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D003587:Cytomegalovirus / Q000235:genetics*; D004622:Embryo, Mammalian; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D005822:Genetic Vectors / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D017919:Hepatitis B Virus, Woodchuck / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D009474:Neurons / Q000378:metabolism* / Q000821:virology; D011401:Promoter Regions, Genetic / Q000502:physiology; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D016704:Synapsins / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D019076:Transgenes
D001555:Benzene Derivatives; D016704:Synapsins; D049452:Green Fluorescent Proteins; C007164:cumene hydroperoxide