title: The use of a viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein (eGFP) in vitro and in vivo.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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 the vgf gene, 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.
  The viral 2A sequence is much smaller than the traditional IRES and therefore allowed over-expression of the vgf gene with fluorescent tracking without compromising viral capacity.
  
  
  CONCLUSIONS
  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.
authors: Division of Nutritional Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK; School of Life Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK.
authors: Jo E
authors: JE
authors: Lewis
authors: Division of Nutritional Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
authors: John M
authors: JM
authors: Brameld
authors: Division of Nutritional Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
authors: Phil
authors: P
authors: Hill
authors: The Rowett Institute of Nutrition and Health, University of Aberdeen, Bucksburn, Aberdeen AB21 9SB, UK.
authors: Perry
authors: P
authors: Barrett
authors: School of Life Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK.
authors: Francis J P
authors: FJ
authors: Ebling
authors: Division of Nutritional Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK. Electronic address: preeti.jethwa@nottingham.ac.uk.
authors: Preeti H
authors: PH
authors: Jethwa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D016384:Consensus Sequence; D004274:DNA, Recombinant; D004279:DNA, Viral; D000229:Dependovirus / Q000235:genetics*; D005821:Genetic Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007031:Hypothalamus / Q000378:metabolism; D008297:Male; D008647:Mesocricetus; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: D004274:DNA, Recombinant; D004279:DNA, Viral; D009414:Nerve Growth Factors; D009479:Neuropeptides; D012333:RNA, Messenger; C512697:Vgf protein, mouse; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Enhanced green fluorescent protein (eGFP); Neuroblastoma SH-SY5Y cells; Recombinant adeno-associated virus (rAAV); Siberian hamster; VGF (non-acronymic); Viral 2A sequence