Abstract
The luciferase reporter gene is a useful tool for determining the efficacy of transfection of plasmid DNA and adenovirus-mediated gene transfer in vivo. However, we report here that the haemoglobin present in tissue samples can mask the detection of the luciferase activity and lead to underestimation of the luciferase levels. We evaluated the degree of interference in different organ samples of mice and investigated the possibilities for removal of haemoglobin from tissue samples by: (1) perfusion of the whole animal; (2) different hypotonic treatments lysing preferentially red blood cells; and (3) chromatographic separation. Removal of haemoglobin resulted in significantly improved detection of luciferase activity from tissue samples. The results indicate that the luciferase activity determined in tissue samples may not reflect the actual level of reporter gene expression, if contaminating blood is not taken into consideration.
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Acknowledgements
This work was supported by grants from the ‘Institut National de la Santé et de la Recherche Médicale’ (INSERM) and from the ‘Association Française de Lutte contre la Mucoviscidose’. We thank Dr A Pavirani of Transgene SA (Strasbourg, France) and Dr M Themis (Imperial College, London) for providing AdMLPLuc and Dr D Gruenert (Gene Therapy Core Center, USA) for the 56FHTE8o− cell line. CC is funded by the Muller Bequest/CF-Trust, the MRC and the March of Dimes Birth Defects Foundation. HS was supported by the Deutsche Forschungsgemeinschaft.
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Colin, M., Moritz, S., Schneider, H. et al. Haemoglobin interferes with the ex vivo luciferase luminescence assay: consequence for detection of luciferase reporter gene expression in vivo. Gene Ther 7, 1333–1336 (2000). https://doi.org/10.1038/sj.gt.3301248
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DOI: https://doi.org/10.1038/sj.gt.3301248
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