The use of viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein eGFP
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. Ne...
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| Format: | Article |
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Elsevier
2015
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| Online Access: | https://eprints.nottingham.ac.uk/29726/ |
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| author | Lewis, Jo E. Brameld, John M. Hill, P.J. Barrett, Perry Ebling, Francis J.P. Jethwa, P.H. |
| author_facet | Lewis, Jo E. Brameld, John M. Hill, P.J. Barrett, Perry Ebling, Francis J.P. Jethwa, P.H. |
| author_sort | Lewis, Jo E. |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | 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. |
| first_indexed | 2025-11-14T19:06:41Z |
| format | Article |
| id | nottingham-29726 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| last_indexed | 2025-11-14T19:06:41Z |
| publishDate | 2015 |
| publisher | Elsevier |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-297262020-05-04T17:14:50Z https://eprints.nottingham.ac.uk/29726/ 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. 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. Elsevier 2015-08-20 Article PeerReviewed Lewis, Jo E., Brameld, John M., Hill, P.J., Barrett, Perry, Ebling, Francis J.P. and Jethwa, P.H. (2015) 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 . pp. 22-29. ISSN 1872-678X Viral 2A sqeuence recombinanat adeno-associated virus (rAAV) Neuroblastoma SHSY-5Y cells VGF Siberian hamsters http://www.sciencedirect.com/science/article/pii/S0165027015003040 doi:10.1016/j.jneumeth.2015.08.013 doi:10.1016/j.jneumeth.2015.08.013 |
| spellingShingle | Viral 2A sqeuence recombinanat adeno-associated virus (rAAV) Neuroblastoma SHSY-5Y cells VGF Siberian hamsters Lewis, Jo E. Brameld, John M. Hill, P.J. Barrett, Perry Ebling, Francis J.P. Jethwa, P.H. The use of viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein eGFP |
| title | The use of viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein eGFP |
| title_full | The use of viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein eGFP |
| title_fullStr | The use of viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein eGFP |
| title_full_unstemmed | The use of viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein eGFP |
| title_short | The use of viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein eGFP |
| title_sort | use of viral 2a sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein egfp |
| topic | Viral 2A sqeuence recombinanat adeno-associated virus (rAAV) Neuroblastoma SHSY-5Y cells VGF Siberian hamsters |
| url | https://eprints.nottingham.ac.uk/29726/ https://eprints.nottingham.ac.uk/29726/ https://eprints.nottingham.ac.uk/29726/ |