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Using a Low LET Electron Microbeam to Investigate Non-Targeted Effects of Low Dose Radiation

Marianne Sowa, Principal Investigator
Bill Morgan, University of Maryland, Co-Principal Investigator

Funding Agency: U.S. Department of Energy, Low Dose Radiation Research Program

Schematic figures of the electron microbeam (A) and the electron microbeam–sample interface (B).
Figure 1. Schematic figures of the electron microbeam (A) and the electron microbeam–sample interface (B). View full image.

A low linear energy transfer (LET) electron microbeam, designed and built at the Pacific Northwest National Laboratory (PNNL) and now housed at the University of Maryland, generates energetic electrons to mimic radiation damage from gamma and X-ray sources. The microbeam has been designed such that high-energy electrons deposit energy in a pre-selected subset of cells, leaving neighboring cells unirradiated (Figure 1). The Using a Low LET Electron Microbeam to Investigate Non-Targeted Effects of Low Dose Radiation project team at PNNL, working in conjunction with co-principal investigator, Bill Morgan, at the Radiation Oncology Research Laboratory, University of Maryland in Baltimore, is using the microbeam to examine non-targeted effects associated with low dose radiation exposure, including induced genomic instability and bystander effects.

Protocol for evaluating induced genomic instability using the GFP reporter assay in RKO36 cells.
Figure 2. Protocol for evaluating induced genomic instability using the GFP reporter assay in RKO36 cells. RKO36 cells contain a mixture of GFP+ (green) and GFP- (clear) cells. They are plated at a low density for colony formation, and instability is evaluated using our automated fluorescence microscope. A colony will be considered stable if all cells are green or all cells are clear and unstable if there is a mixture of GFP+ and GFP- cells in the colony. Our usual criteria for instability is >5 cells of one cell type (e.g., GFP+) in a colony containing predominantly GFP- cells. View full image.

This research takes advantage of a high-throughput assay for hyper-recombination and deletion events. This assay was developed during the current funding period of our U.S. Department of Energy (DOE) grant and uses a novel green fluorescence protein (GFP) reporter assay. A summary of the experimental strategy for determining delayed radiation-induced genomic instability is presented in Figure 2. Analysis of stable (GFP- or GFP+) colonies and unstable (GFP+/-) colonies has been fully automated using fluorescence microscopy and provides a robust, reliable, highly sensitive assay for detecting delayed events occurring in cells exposed to low doses of ionizing radiation.

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