Office of Biological and Environmental Research

DOE Low Dose Radiation Program Workshop V

2005 Abstract

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

Authors: Katherine A Vallis,^1,2,3 William L Stanford,⁴ Zhuo Chen,³ Lindsay Park,³ Jorge Wong,³ Mandeep Grewal,³ and Sherri Liu³

Institutions: ¹Departments of Radiation Oncology and ²Medical Biophysics, University of Toronto and the ³Princess Margaret Hospital/Ontario Cancer Institute, ⁴Institute of Biomaterials and Biomedical Engineering, University of Toronto

We are conducting a 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. The DNA vector used in this system is the PT1-ATG vector, which consists of a splice acceptor site immediately upstream of a promoterless LacZ reporter gene. In addition, this vector contains the bacterial neomycin resistance (neoR) gene driven by an autonomous phosphoglycerate kinase-1 (PGK-1) promoter, which serves as a selectable marker. The integration of this vector into an intronic element leads to the generation of a fusion transcript between the endogenous gene (into which the vector has inserted) and the LacZ reporter upon transcriptional activation of the endogenous gene. LacZ thus acts as an accurate readout for transcriptional activation of the trapped gene. Differences in x-gal staining between irradiated and control cultures allow the identification of positive clones, in which the trapped gene is regulated by ionizing radiation (IR). The locus in which the gene trap vector inserts can be identified by 5'-rapid amplification of cDNA ends (RACE). Interesting gene trap clones are selected based on their sequence identity, the magnitude of induction or down-regulation following IR, and their potential for generating null mutations. The advantage of the gene trap system is that it is possible to mutate the trapped gene while still reporting its normal expression pattern in response to IR. Since the screen is conducted in murine embryonic stem (ES) cells, germ line transmission of the gene trap mutation and study of gene expression in the context of the intact animal, are possible.

We have screened 4,992 gene trap clones for responsiveness to both moderate dose (3 Gy; ¹³⁷Cs, dose rate 1.14 Gy/min) and low dose (10 cGy; ⁶⁰Co, dose rate, 1.7 cGy/min) IR. Of these, 427 (8.6%) clones expressed LacZ. We scored the intensity of x-gal staining using a 5-point scale (0, none; 1, barely detectable staining; 2, modest but easily detectable staining; 3, moderate; 4, strong). We also scored the percentage of cells that stained for x-gal according to a 6-point scale (0, 0%; 1, <1%; 2, 1-10% ; 3, 11-33%; 4, 34-66%; 5, 67-95%; 6, >95%). In 98 clones there was a 1 or 2 level difference in either the intensity of x-gal staining, in the percentage of cells that expressed LacZ, or both, in irradiated compared to non-irradiated, control cultures. Staining was repeated in these cases, to confirm or refute the result from the initial screen. In this system, responsiveness to LDR was infrequent compared to moderate dose IR. Six clones showed a consistent alteration in LacZ expression following LDR and two (designated 17D5 and 20H5) have been selected for in-depth analyses. Sequencing of the 5'-RACE product of the 17D5 gene trap clone identified the LacZ insertion locus as POLN, a recently identified nuclear PolA family DNA polymerase (Accession number: AY135562). A possible role for the POLN polymerase subfamily in DNA repair has recently been suggested. POLN is highly expressed in human and mouse testis, also suggesting a potential role for POLN in meiosis. Sequencing of the 5'-RACE product of the 20H5 gene trap clone identified the LacZ insertion locus as ELOVL family member 6 (ELOVL6; Accession number: NM_130450). This gene encodes a lipogenic enzyme regulated by sterol regulatory element-binding protein 1 (SREBP-1) and plays a role in de novo synthesis of long-chain saturated and monosaturated fatty acids. In vitro expression analyses to confirm IR-regulation of these genes have been conducted and we now plan to generate mutant mice, using the 17D5 and 20H5 gene trap clones, for in vivo expression analyses of these genes in response to LDR.

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

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