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Visualizing protein-protein interactions in living animals
Gary D Luker1, Vijay Sharma, David Piwnica-Worms
1Department of Molecular Biology, Molecular Imaging Center, Mallinckrodt Institute of Radiology, Washington University Medical School, 510 South Kingshighway Boulevard, 63110, St. Louis, MO, USA.
Abstract:
A variety of techniques have been developed to analyze protein-protein interactions in vitro and in cultured cells. However, these methods do not determine how protein interactions affect and are regulated by physiologic and pathophysiologic conditions in living animals. This article describes methodology for detecting and quantifying protein interactions in living mice, using an inducible two-hybrid system developed for positron emission tomography (PET) imaging. We discuss the methods to establish stably transfected cells with components of the imaging system, create tumor xenografts, synthesize PET radiopharmaceuticals used to visualize the imaging reporter, perform microPET imaging, and analyze data from imaging studies. Development and application of technologies for molecular imaging of protein-protein interactions in vivo should enable researchers to investigate intrinsic binding specificities of proteins during normal development and disease progression as well as aid drug development through direct interrogation of molecular targets within intact animals.
Insights
This study introduces a novel positron emission tomography (PET) imaging method to track protein-protein interactions in living mice. This technique allows researchers to study these interactions in vivo during disease and development.
Area of Science:
- Molecular Biology
- Biochemistry
- Medical Imaging
Background:
- Existing methods for studying protein-protein interactions are limited to in vitro and cell culture settings.
- These methods fail to capture how physiological and pathophysiological conditions in living animals regulate these interactions.
- There is a need for in vivo methodologies to assess protein interactions within their native biological context.
Purpose of the Study:
- To describe a methodology for detecting and quantifying protein-protein interactions in living mice.
- To adapt an inducible two-hybrid system for positron emission tomography (PET) imaging.
- To enable the study of protein interactions in vivo under various physiological and pathological conditions.
Main Methods:
- Establishment of stably transfected cells expressing components of the inducible two-hybrid system.
- Creation of tumor xenografts for in vivo studies.
- Synthesis of PET radiopharmaceuticals for imaging reporter visualization.
- Performance of microPET imaging and subsequent data analysis.
Main Results:
- Successful development and implementation of a PET-based imaging system for in vivo protein-protein interaction analysis.
- Demonstration of the methodology's capability to detect and quantify protein interactions in living animals.
- Validation of the system for use in tumor xenograft models.
Conclusions:
- The described methodology enables molecular imaging of protein-protein interactions in living mice.
- This technology facilitates the investigation of protein binding specificities during normal development and disease progression.
- The system aids drug development by allowing direct interrogation of molecular targets within intact animals.