Research Project
6795657
DESCRIPTION (provided by applicant): The green fluorescent protein (GFP) from Aequorea victoria has been developed into a highly versatile reporter gene. Another family of fluorescent proteins, including Discosoma Red (DsRed), has been cloned from corals (4). This was followed by Anemonia asRed (5) and HcRed (6). We have developed GFP as an in vivo whole-body imaging reporter for cancer and metastasis, gene expression, and infectious disease. In these applications, the red emission is of special importance for minimizing background emission and scattering in vivo as well as for FRET (fluorescence resonance energy transfer) analysis. High extinction coefficients, quantum yield and the monomeric state of proteins are very important parameters for the above-mentioned applications. In contrast to GFP, which has only a small tendency to dimerize, the new proteins from corals have a pronounced tendency to form oligomers, e.g. tetramers are observed for DsRed, or even higher aggregates (11,12). Extinction coefficients and quantum yields are also relatively low for red proteins (4-6) and for the newly developed monomeric DsRed (17). Thus, there is an important need for new reporters that are monomeric, and therefore good candidates for fusion to other genes with high extinction coefficients and long wavelength emission for in vivo use. Discovery of novel fluorescent proteins has previously used potential sequence and structural homology of beta-strands or stretches between beta-strands of novel proteins with those of GFP from A. victoria (4- 6). These primers, used to clone the new fluorescence proteins, code for surface amino acids. Despite the significant success of this strategy in discovery of novel fluorescent proteins in corals and related organisms, the approach based on sequence homology has serious limitations. To overcome this limitation, a discovery strategy initially based on separation of candidate proteins from selected marine organisms, by 2-D electrophoresis and then analyzed by fluorescence spectroscopy will be developed. The first step is to localize novel fluorescent proteins from murine organisms by spectral and protein analysis of the total body of the marine organism. After separation of candidate proteins by 2-D gel electrophresis, and fluorescence spectroscopy, the proteins will be sequenced and cloned by novel primer design and PCR. The second step is to generate variants of the reporters by mutagenesis of the novel proteins. The goal is to identify novel fluorescent proteins with emission peaks greater than 620 nm, high extinction coefficients, and high quantum yields. Thorough evaluation of the novel fluorescent proteins will be carried out by whole-body imaging in mice with tumor cells expressing these genes using an orthotopic model of lung cancer growing on the lung and metastasized to other organs. Characterization will also include immunogenicity and proteolytic stability of the new fluorescent protein. Reporters with spectral properties that can be used for whole-body imaging of tumors in the lung and their metastases will be candidates for further development for numerous applications of multi-color imaging.
