Human gene therapy vol:19 issue:10 pages:1129-1130
European Society of Gene & Cell Therapy location:Brugge, Belgium date:13-16 November 2008
Background: The transgenic overexpression of proteins in vivo is a powerful approach to study the basic biology of proteins, to generate disease models or to evaluate gene therapy approaches. When investigating a transgenic protein, a specific and sensitive detection is indispensable. Unfortunately, antibodies raised against the protein of interest are not always available. It is also necessary to discriminate endogenous from overexpressed proteins. The use of an epitope tag fused to the protein may circumvent these difficulties. In order to minimize impact on the bioactivity and biodistribution of the overexpressed protein, preference is given to small tags.
In the present study, we evaluated several small epitope tag/antibody combinations for the detection of overepxressed proteins in cell extracts as well as in intact rodent brain, using eGFP as a reference.
Methods: We fused different epitope tags (AU1, flag, 3flag, HA, myc and V5) N-terminally to eGFP and made lentiviral vectors with these constructs. After transduction of HEK293T cells, we analysed the different constructs via SDS-PAGE. Using the same lentivectors, stereotaxic injections were performed in the striatum of Wistar rats. After staining the vibratome sections with the appropriate tag antibodies, we examined the size of the transduced area and the number of transduced cells recognised by the tag antibody compared to eGFP detection.
Results: We found that all the examined antibodies can detect the tag-eGFP fusion protein in cell extracts as well as in vivo, in varying degrees. The anti-V5 antibody seems to detect the V5-eGFP in a very sensitive manner, both on western blotting and in vivo. Currently, we are further investigating the sensitivity and specificity of these epitope tag/antibody combinations in vivo.
Discussion & conclusion: The selection of an appropriate epitope tag provides the possibility to have an unambiguous detection of any overexpressed gene of interest.