ToxSci Advance Access originally published online on February 23, 2009
Toxicological Sciences 2009 109(1):96-105; doi:10.1093/toxsci/kfp037
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Published by Oxford University Press 2009.
The Mouse Lymphoma Assay Detects Recombination, Deletion, and Aneuploidy
* Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research/ Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079
Cytogenetics Laboratory, Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR 72205
College of Life Science and Technology, Shanghai Jiao Tong University, Shanghai, China
Division of Genetics and Mutagenesis, National Institute of Health Sciences, Tokyo 158-8501, Japan
2 To whom correspondence should be addressed at Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research/FDA, 3900 NCTR Rd, Jefferson, AR 72079. Fax: 870-543-7393. E-mail: martha.moore{at}fda.hhs.gov.
Received October 31, 2008; accepted January 29, 2009
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Abstract |
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The mouse lymphoma assay (MLA) uses the thymidine kinase (Tk) gene of the L5178Y/Tk+/–-3.7.2C mouse lymphoma cell line as a reporter gene to evaluate the mutagenicity of chemical and physical agents. The MLA is recommended by both the United States Food and Drug Administration and the United States Environmental Protection Agency as the preferred in vitro mammalian cell mutation assay for genetic toxicology screening because it detects a wide range of genetic alterations, including both point mutations and chromosomal mutations. However, the specific types of chromosomal mutations that can be detected by the MLA need further clarification. For this purpose, three chemicals, including two clastogens and an aneugen (3'-azido-3'-deoxythymidine, mitomycin C, and taxol), were used to induce Tk mutants. Loss of heterozygosity (LOH) analysis was used to select mutants that could be informative as to whether they resulted from deletion, mitotic recombination, or aneuploidy. A combination of additional methods, G-banding analysis, chromosome painting, and a real-time PCR method to detect the copy number (CN) of the Tk gene was then used to provide a detailed analysis. LOH involving at least 25% of chromosome 11, a normal karyotype, and a Tk CN of 2 would indicate that the mutant resulted from recombination, whereas LOH combined with a karyotypically visible deletion of chromosome 11 and a Tk CN of 1 would indicate a deletion. Aneuploidy was confirmed using G-banding combined with chromosome painting analysis for mutants showing LOH at every microsatellite marker on chromosome 11. From this analysis, it is clear that mouse lymphoma Tk mutants can result from recombination, deletion, and aneuploidy.
Key Words: mouse lymphoma assay; mutation type; loss of heterozygosity; cytogenetics; copy number; 3'-azido-3'-deoxythymidine; mitomycin C; taxol; thymidine kinase.
1 Present address: Office of New Drugs, Center for Drug Evaluation and Research, Food and Drug Administration, HFD-540, 10903 New Hampshire Avenue, Silver Spring, MD 20993.