Office of Biological and Environmental Research

DOE Low Dose Radiation Program Workshop IV

Abstract

Title: The Characterization of Genetic Responses to Low Dose Radiation using a Genome-Wide Insertional Mutagenesis Approach

Authors: Katherine A. Vallis¹ and William L. Stanford²

Institutions: ¹ Departments of Radiation Oncology and Medical Biophysics, University of Toronto and the Princess Margaret Hospital, ² Institute of Biomaterials and Biomedical Engineering, University of Toronto

We have recently initiated a high-throughput, genome-wide screen to identify mammalian genes that are regulated by low dose radiation (LDR). We are using a gene trap mutagenesis approach to do this. In this system a mutagenizing DNA vector, introduced into cells by electroporation, disrupts genes randomly. The gene trap vector contains a splice acceptor site upstream of a promoterless reporter (lacZ). On transcriptional activation of the endogenous promoter of the trapped gene, a fusion transcript is generated from the upstream coding sequence and the reporter gene, simultaneously mutating the trapped gene and reporting its expression pattern. The vector also contains the selectable neo gene driven by an autonomous promoter. The fusion transcript acts as a tag for identification of the gene, by cloning the fusion mRNA and using RT-PCR techniques. We have conducted such a screen using moderate dose radiation (2 to 4 Gy, ¹³⁷Cs, dose rate 1.14 Gy/minute) and have demonstrated this to be a robust approach to the study of genetic radiation responses. We propose now to carry out a similar screen over the next two years, using 10 cGy as the screening dose (⁶⁰Co; dose rate 1.7 cGy/minute) to evaluate the utility of this approach in the study of genetic responses to LDR. Since we will apply the screen to murine embryonic stem (ES) cells, we will be able to generate mutant animals by introducing ES gene trap cell lines of interest into the mouse germ line. These animals will allow us to study the in-vivo expression pattern of the gene of interest in control and low dose irradiated animals. Importantly, we can study tissue- and cell-specificity of expression of the gene of interest, which is "tagged" with the reporter lacZ, in the intact animal. We will use cDNA microarray analysis as a complementary functional genomics approach to corroborate LDR-responsiveness of genes of interest, to identify co-regulated genes, and to identify other LDR responsive genes that may be displayed on the array.

(U.S. Department of Energy, Grant No DE-FG02-03ER63644)

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