Molecular Cloning and Expression of 6HIS - GFP Reporter Gene for Fluorescent and Electron Spectroscopic Imaging.

Abstract

Molecular cloning offers an opportunity for designing constructs for expressing chimeric proteins containing incorporated reporter molecules. In this approach, molecules are identified based upon reporter molecules which are expressed within cells as a result of transfection, instead of marking them through immunolabeling, in situ hybridization, or derivative incorporation. This approach is particularly useful for studies involving integrated microscopy. Integrated microscopy allows us to assemble images of the same cell obtained with different microscopes into one comprehensive message concerning cellular functions. Integration of fluorescence and electron spectroscopic imaging is particularly promising. The main advantage of this approach relies in overcoming limitations of each type of microscopy alone i.e. in studies on living cells limitations in spatial resolution of light microscopy and in analysis of supramolecular organization, limitation of electron microscopy to frozen or fixed cells. For this purpose, special reporter molecules suitable for selected modes of microscopy have to be used.