Molecular Imaging of Hypoxia Using Genetic Biosensors
- 1.1k Downloads
In the last years, the need for visualization of tumoral processes has become a high-top priority in molecular imaging. This is especially true for those methods dedicated to functional imaging that focus on revealing phenomena associated with biological processes such as hypoxia to cancer. Among them, optical imaging methods such as fluorescence are provided with a broad range of proteins and dyes used to visualize many types of these biological processes widely used in cell biology studies. Although the most popular of these proteins is the green fluorescent protein (GFP), autofluorescence due to the absorption of the exciting radiation by endogenous fluorophores and signal dispersion raises doubts about its suitability as an in vivotracer. In the last years a number of groups have developed several NIR fluorescent proteins that enables real-time imaging to take place without interference from autofluorescence events allowing at the same time to take a deep view into the tissues. With all of this in mind, we devised a novel fluorescence-bioluminescence genetically encoded biosensor activated by the neoangiogenesis-related transcription factor HIF-1α, which appears upregulated in growing tumors. At the same time, by fusing a NIR emitting flurochrome (mCherry) and the firefly luciferase together we obtained a bioluminescence resonance energy transfer (BRET) phenomenon turning this fusion protein into a new class of hypoxia-sensing genetically encoded biosensor.
We thank the members of Molecular Oncology Lab for helpful discussions. We also thank M.E. Vazquez for helpful discussions and assistance with the spectrophotometric analysis. This study was supported by Spanish Ministry of Science and Innovation, (SAF2005-00306; SAF2008-00543) and Xunta de Galicia grants (PGIDIT05PXIB20801PR); Grupos emerxentes 2007/064) (J.A.C.), and by Fundacion de Investigacion Medica Mutua Madrileña (J.A.C., P.I.).
- 2.Akagi, Y., et al.: Regulation of vascular endothelial growth factor expression in human colon cancer by insulin-like growth factor-I. Cancer Res. 58, 4008–4014 (1998)Google Scholar
- 13.Hoffman, R.M.: Imaging tumor angiogenesis with fluorescent proteins. APMIS 112, 441–449 (2004)Google Scholar
- 19.Ntziachristos, V., Bremer, C., Weissleder, R.: Fluorescence imaging with near-infrared light: new technological advances that enable in vivo molecular imaging. Eur. Radiol. 13, 195–208 (2003)Google Scholar
- 25.Shaner, N.C., Campbell, R.E., Steinbach, P.A., etal.: Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572(2004)Google Scholar
- 26.Shaner, N.C., Steinbach, P.A., Tsien, R.Y.: A guide to choosing fluorescent proteins. Nature 2, 905–909(2005)Google Scholar