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The use of viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein eGFP

Lewis, Jo E.; Brameld, John M.; Hill, P.J.; Barrett, Perry; Ebling, Francis J.P.; Jethwa, P.H.

Authors

Jo E. Lewis

JOHN BRAMELD JOHN.BRAMELD@NOTTINGHAM.AC.UK
Professor of Nutritional Biochemstry

P.J. Hill

Perry Barrett

Francis J.P. Ebling fran.ebling@nottingham.ac.uk



Abstract

Introduction: The viral 2A sequence has become an attractive alternative to the traditional internal ribosomal entry site (IRES) for simultaneous over-expression of two genes and in combination with recombinant adeno-associated viruses (rAAV) has been used to manipulate gene expression in vitro.
New Method: To develop a rAAV construct in combination with the viral 2A sequence to allow long-term over-expression of the vgf gene and fluorescent marker gene for tracking of the transfected neurones in vivo.
Results: Transient transfection of the AAV plasmid containing the vgf gene, viral 2A sequence and eGFP into SH-SY5Y cells resulted in eGFP fluorescence comparable to a commercially available reporter construct. This increase in fluorescent cells was accompanied by an increase in VGF mRNA expression. Infusion of the rAAV vector containing VGF, viral 2A sequence and eGFP resulted in eGFP fluorescence in the hypothalamus of both mice and Siberian hamsters, 32 weeks post infusion. In-situ hybridisation confirmed that the location of VGF mRNA expression in the hypothalamus corresponded to the eGFP pattern of fluorescence.
Comparison with old method: The viral 2A sequence is much smaller than the traditional IRES and therefore allowed over-expression of vgf with fluorescent tracking without compromising viral capacity.
Conclusion: The use of the viral 2A sequence in the AAV plasmid allowed the simultaneous expression of both genes in vitro. When used in combination with rAAV it resulted in long-term over-expression of both genes at equivalent locations in the hypothalamus of both Siberian hamsters and mice, without any adverse effects.

Journal Article Type Article
Journal Journal of Neuroscience Methods
Print ISSN 0165-0270
Electronic ISSN 1872-678X
Publisher Elsevier
Peer Reviewed Peer Reviewed
Volume 256
APA6 Citation Lewis, J. E., Brameld, J. M., Hill, P., Barrett, P., Ebling, F. J., & Jethwa, P. (in press). The use of viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein eGFP. Journal of Neuroscience Methods, 256, https://doi.org/10.1016/j.jneumeth.2015.08.013
DOI https://doi.org/10.1016/j.jneumeth.2015.08.013
Keywords Viral 2A sqeuence
recombinanat adeno-associated virus (rAAV)
Neuroblastoma SHSY-5Y cells
VGF
Siberian hamsters
Publisher URL http://www.sciencedirect.com/science/article/pii/S0165027015003040
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0

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Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0





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