Discovery of novel fluorescent reporter genes

Summary

Principal Investigator: R M Hoffman
Abstract: 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.
Funding Period: 2004-04-01 - 2005-03-31
more information: NIH RePORT

Top Publications

  1. ncbi Hair follicle-derived blood vessels vascularize tumors in skin and are inhibited by Doxorubicin
    Yasuyuki Amoh
    AntiCancer, Inc, San Diego, CA 92111 3604, USA
    Cancer Res 65:2337-43. 2005
  2. pmc Dual-color imaging of angiogenesis and its inhibition in bone and soft tissue sarcoma
    Katsuhiro Hayashi
    Department of Surgery, University of California San Diego, San Diego, California, USA
    J Surg Res 140:165-70. 2007
  3. ncbi In vivo color-coded imaging of the interaction of colon cancer cells and splenocytes in the formation of liver metastases
    Michael Bouvet
    Department of Surgery, University of California, San Diego, USA
    Cancer Res 66:11293-7. 2006
  4. ncbi Visualization of nascent tumor angiogenesis in lung and liver metastasis by differential dual-color fluorescence imaging in nestin-linked-GFP mice
    Yasuyuki Amoh
    AntiCancer, Inc, 7917 Ostrow Street, San Diego, CA 92111, USA
    Clin Exp Metastasis 23:315-22. 2006
  5. ncbi Tumor cells genetically labeled with GFP in the nucleus and RFP in the cytoplasm for imaging cellular dynamics
    Ping Jiang
    AntiCancer, Inc, San Diego, California 92111, USA
    Cell Cycle 5:1198-201. 2006
  6. ncbi Development of real-time subcellular dynamic multicolor imaging of cancer-cell trafficking in live mice with a variable-magnification whole-mouse imaging system
    Kensuke Yamauchi
    AntiCancer, Inc, San Diego, California 92111, USA
    Cancer Res 66:4208-14. 2006
  7. ncbi Common bile duct injection as a novel method for establishing red fluorescent protein (RFP)-expressing human pancreatic cancer in nude mice
    Kazuhiko Tsuji
    Department of Surgery, University of California San Diego, San Diego, CA, USA
    JOP 7:193-9. 2006
  8. ncbi Dual-color imaging of nascent blood vessels vascularizing pancreatic cancer in an orthotopic model demonstrates antiangiogenesis efficacy of gemcitabine
    Yasuyuki Amoh
    Department of Surgery, University of California San Diego, San Diego, California, USA
    J Surg Res 132:164-9. 2006
  9. ncbi Dual-color imaging of nuclear-cytoplasmic dynamics, viability, and proliferation of cancer cells in the portal vein area
    Kazuhiko Tsuji
    AntiCancer, Inc, San Diego, California 92111, USA
    Cancer Res 66:303-6. 2006
  10. ncbi In vivo cell biology of cancer cells visualized with fluorescent proteins
    Robert M Hoffman
    AntiCancer, Inc, San Diego, CA 92111, USA
    Curr Top Dev Biol 70:121-44. 2005

Scientific Experts

  • Y Amo
  • Robert M Hoffman
  • Yasuyuki Amoh
  • Michael Bouvet
  • Kazuhiko Tsuji
  • Kensuke Yamauchi
  • Ping Jiang
  • Meng Yang
  • Abdool R Moossa
  • Kensei Katsuoka
  • Lingna Li
  • Katsuro Tomita
  • Norio Yamamoto
  • Hiroyuki Tsuchiya
  • A R Moossa
  • Mingxu Xu
  • Katsuhiro Hayashi
  • Anirban Maitra
  • Matthew H Katz
  • Olga Berezovskaya
  • Yoshikatsu Kanai
  • Hitoshi Endo
  • Koji Yamashita
  • Sharmeela Kaushal
  • Ralph Bernacki
  • Gennadi V Glinsky
  • John C Reed
  • Richard V Mazurchuk
  • Aaron D Schimmer
  • Clemencia Pinilla
  • Joseph Spernyak
  • Anna B Glinskii
  • Youcef M Rustum
  • Shinako Takimoto
  • Raul Campillo
  • Sheldon Penman
  • Jose Reynoso
  • Katsumasa Kawahara

Detail Information

Publications16

  1. ncbi Hair follicle-derived blood vessels vascularize tumors in skin and are inhibited by Doxorubicin
    Yasuyuki Amoh
    AntiCancer, Inc, San Diego, CA 92111 3604, USA
    Cancer Res 65:2337-43. 2005
    ..Doxorubicin inhibited the nascent tumor angiogenesis as well as tumor growth in the ND-GFP mice transplanted with B16F10-RFP. This model is useful for direct visualization of tumor angiogenesis and evaluation of angiogenic inhibitors...
  2. pmc Dual-color imaging of angiogenesis and its inhibition in bone and soft tissue sarcoma
    Katsuhiro Hayashi
    Department of Surgery, University of California San Diego, San Diego, California, USA
    J Surg Res 140:165-70. 2007
    ..We demonstrate in this report, using powerful color-coded fluorescent imageable tumor-host models, the onset of angiogenesis of these sarcomas and its inhibition...
  3. ncbi In vivo color-coded imaging of the interaction of colon cancer cells and splenocytes in the formation of liver metastases
    Michael Bouvet
    Department of Surgery, University of California, San Diego, USA
    Cancer Res 66:11293-7. 2006
    ..These results suggest a novel tumor-host interaction that enables efficient formation of liver metastasis via intrasplenic injection...
  4. ncbi Visualization of nascent tumor angiogenesis in lung and liver metastasis by differential dual-color fluorescence imaging in nestin-linked-GFP mice
    Yasuyuki Amoh
    AntiCancer, Inc, 7917 Ostrow Street, San Diego, CA 92111, USA
    Clin Exp Metastasis 23:315-22. 2006
    ..The model will serve as a unique screen for inhibitors of angiogenesis of metastatic tumors. The fact that liver-metastasis angiogenesis can be imaged in the live animal enables real-time studies of the effect of angiogenesis inhibitors...
  5. ncbi Tumor cells genetically labeled with GFP in the nucleus and RFP in the cytoplasm for imaging cellular dynamics
    Ping Jiang
    AntiCancer, Inc, San Diego, California 92111, USA
    Cell Cycle 5:1198-201. 2006
    ..The dual-color cells described here thus enable the sub-cellular dynamics of cancer cell trafficking to be imaged in the living animal...
  6. ncbi Development of real-time subcellular dynamic multicolor imaging of cancer-cell trafficking in live mice with a variable-magnification whole-mouse imaging system
    Kensuke Yamauchi
    AntiCancer, Inc, San Diego, California 92111, USA
    Cancer Res 66:4208-14. 2006
    ..This imaging technology will enable further understanding of the critical steps of metastasis and provide visible targets for antimetastasis drug development...
  7. ncbi Common bile duct injection as a novel method for establishing red fluorescent protein (RFP)-expressing human pancreatic cancer in nude mice
    Kazuhiko Tsuji
    Department of Surgery, University of California San Diego, San Diego, CA, USA
    JOP 7:193-9. 2006
    ..In our previous pancreatic cancer mouse models, we have used surgical orthotopic implantation of human pancreatic tumors to establish clinically relevant fluorescent mouse models of pancreatic cancer...
  8. ncbi Dual-color imaging of nascent blood vessels vascularizing pancreatic cancer in an orthotopic model demonstrates antiangiogenesis efficacy of gemcitabine
    Yasuyuki Amoh
    Department of Surgery, University of California San Diego, San Diego, California, USA
    J Surg Res 132:164-9. 2006
    ..The stem cell marker nestin recently has been shown to be expressed in nascent blood vessels in nestin-driven green fluorescent protein (ND-GFP) transgenic nude mice...
  9. ncbi Dual-color imaging of nuclear-cytoplasmic dynamics, viability, and proliferation of cancer cells in the portal vein area
    Kazuhiko Tsuji
    AntiCancer, Inc, San Diego, California 92111, USA
    Cancer Res 66:303-6. 2006
    ..These results suggest that a cyclophosphamide-sensitive host cellular system attacked the HCT-116-GFP-RFP cells but could not effectively kill the MMT-GFP-RFP cells...
  10. ncbi In vivo cell biology of cancer cells visualized with fluorescent proteins
    Robert M Hoffman
    AntiCancer, Inc, San Diego, CA 92111, USA
    Curr Top Dev Biol 70:121-44. 2005
    ..The use of the dual-color cancer cells differentially labeled in the cytoplasm and nucleus and associated fluorescent imaging provide a powerful tool to understand the mechanism of cancer cell migration and deformation in small vessels...
  11. pmc Implanted hair follicle stem cells form Schwann cells that support repair of severed peripheral nerves
    Yasuyuki Amoh
    AntiCancer, Inc, 7917 Ostrow Street, San Diego, CA 92111, USA
    Proc Natl Acad Sci U S A 102:17734-8. 2005
    ..These results suggest that hair follicle stem cells provide an important, accessible, autologous source of adult stem cells for regenerative medicine...
  12. ncbi High correlation of whole-body red fluorescent protein imaging and magnetic resonance imaging on an orthotopic model of pancreatic cancer
    Michael Bouvet
    Department of Surgery, University of California at San Diego, CA 92111, USA
    Cancer Res 65:9829-33. 2005
    ..FPI has high resolution and is exceedingly rapid with instant image capture. We suggest a complimentary role for these two imaging modalities...
  13. ncbi Nestin-linked green fluorescent protein transgenic nude mouse for imaging human tumor angiogenesis
    Yasuyuki Amoh
    AntiCancer, Inc, San Diego, California 92111, USA
    Cancer Res 65:5352-7. 2005
    ..These results suggest that this model is useful for the imaging of the angiogenesis of human as well as rodent tumors and visualization of the efficacy of angiogenetic inhibitors...
  14. ncbi Real-time in vivo dual-color imaging of intracapillary cancer cell and nucleus deformation and migration
    Kensuke Yamauchi
    AntiCancer, Inc, San Diego, California 92111, USA
    Cancer Res 65:4246-52. 2005
    ..The use of the dual-color cancer cells differentially labeled in the cytoplasm and nucleus and associated fluorescent imaging provide a powerful tool to understand the mechanism of cancer cell migration and deformation in small vessels...
  15. ncbi Increased expression of apoptosis inhibitor protein XIAP contributes to anoikis resistance of circulating human prostate cancer metastasis precursor cells
    Olga Berezovskaya
    The Sidney Kimmel Cancer Center, San Diego, CA 92121, USA
    Cancer Res 65:2378-86. 2005
    ..We also provide initial preclinical data on the antimetastatic efficacy of recently discovered small-molecule antagonists of XIAP...
  16. ncbi Color-coded fluorescent protein imaging of angiogenesis: the AngioMouse models
    Yasuyuki Amoh
    Department of Dermatology, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan
    Curr Pharm Des 14:3810-9. 2008
    ..These fluorescent models, generally termed AngioMouse, can quantitatively determine efficacy of antiangiogenesis compounds...