Noninvasive quantitative imaging of protein–protein interactions in living subjects

We are developing methods to image molecular and cellular events in living subjects. In this study, we validate imaging of protein—protein interactions in living mice by using bioluminescent optical imaging. We use the well studied yeast two-hybrid system adapted for mammalian cells and modify it to be inducible. We employ the NF-κB promoter to drive expression of two fusion proteins (VP16-MyoD and GAL4-ID). We modulate the NF-κB promoter through tumor necrosis factor α. Firefly luciferase reporter gene expression is driven by the interaction of MyoD and ID through a transcriptional activation strategy. We demonstrate the ability to detect this induced protein–protein interaction in cell culture and image this induced interaction in living mice by using transiently transfected cells. The current approach will be a valuable and potentially generalizable tool to noninvasively and quantitatively image protein–protein interactions in living subjects. The approaches validated should have important implications for the study of protein–protein interactions in cells maintained in their natural in vivo environment as well as for the in vivo evaluation of new pharmaceuticals targeted to modulate protein–protein interactions.

[1]  Krzysztof P Bobinski,et al.  Seeing is believing: Non‐invasive, quantitative and repetitive imaging of reporter gene expression in living animals, using positron emission tomography , 2000, Journal of neuroscience research.

[2]  S S Gambhir,et al.  Noninvasive optical imaging of firefly luciferase reporter gene expression in skeletal muscles of living mice. , 2001, Molecular therapy : the journal of the American Society of Gene Therapy.

[3]  Harold Weintraub,et al.  The protein Id: A negative regulator of helix-loop-helix DNA binding proteins , 1990, Cell.

[4]  S. Fields,et al.  A novel genetic system to detect protein–protein interactions , 1989, Nature.

[5]  David K. Stevenson,et al.  Bioluminescent indicators in living mammals , 1998, Nature Medicine.

[6]  H. Shimada,et al.  Widespread skeletal metastatic potential of human lung cancer revealed by green fluorescent protein expression. , 1998, Cancer research.

[7]  C. Contag,et al.  Use of reporter genes for optical measurements of neoplastic disease in vivo. , 2000, Neoplasia.

[8]  S. Gambhir,et al.  Optical imaging of Renilla luciferase reporter gene expression in living mice , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[9]  A. Saltzman,et al.  hUBC9 associates with MEKK1 and type I TNF‐α receptor and stimulates NFκB activity , 1998 .

[10]  Robert K. Davis,et al.  The myoD gene family: nodal point during specification of the muscle cell lineage. , 1991, Science.

[11]  S. Elledge,et al.  Gene identification using the yeast two-hybrid system. , 1997, Methods in enzymology.

[12]  S. Cherry,et al.  Repetitive, non-invasive imaging of the dopamine D2 receptor as a reporter gene in living animals , 1999, Gene Therapy.

[13]  L. O’Neill,et al.  Ceramide Activates NFκB by Inducing the Processing of p105* , 1998, The Journal of Biological Chemistry.

[14]  J. Humm,et al.  Imaging herpes virus thymidine kinase gene transfer and expression by positron emission tomography. , 1998, Cancer research.

[15]  S. Cherry,et al.  Imaging of adenoviral-directed herpes simplex virus type 1 thymidine kinase reporter gene expression in mice with radiolabeled ganciclovir. , 1998, Journal of nuclear medicine : official publication, Society of Nuclear Medicine.

[16]  M. Iyer,et al.  Two-step transcriptional amplification as a method for imaging reporter gene expression using weak promoters , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[17]  C. Dang,et al.  Detection and modulation in vivo of helix-loop-helix protein-protein interactions. , 1993, The Journal of biological chemistry.

[18]  S. Cherry,et al.  Imaging adenoviral-directed reporter gene expression in living animals with positron emission tomography. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[19]  J. Barrio,et al.  Monitoring gene therapy with reporter gene imaging. , 2001, Seminars in nuclear medicine.

[20]  J. Barrio,et al.  Assays for noninvasive imaging of reporter gene expression. , 1999, Nuclear medicine and biology.

[21]  H. Blau,et al.  Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[22]  A. Varshavsky,et al.  Split ubiquitin as a sensor of protein interactions in vivo. , 1994, Proceedings of the National Academy of Sciences of the United States of America.