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Characterizing Benzo[a]pyrene-induced lacZ mutation spectrum in transgenic mice using next-generation sequencing
Marc A Beal1,2, Rémi Gagné3, Andrew Williams4
1Carleton University, Ottawa, ON, K1S 5B6, Canada. marc.beal@hc-sc.gc.ca.
BMC Genomics
|October 21, 2015
Summary
Next-generation sequencing (NGS) enables high-throughput analysis of mutagenicity using the transgenic rodent mutation reporter assay. This faster, more sensitive method improves the evaluation of genotoxins and reveals new mutation hotspots.
Area of Science:
- Genomics
- Toxicology
- Molecular Biology
Background:
- The transgenic rodent mutation reporter assay is valuable for identifying mutagens in vivo.
- Sequencing mutant transgenes reveals mutagen mechanisms and clonal expansion.
- Conventional sequencing is laborious and costly for long transgenes like lacZ.
Purpose of the Study:
- To develop a high-throughput next-generation sequencing (NGS) method for analyzing large numbers of barcoded mutant lacZ transgenes.
- To apply this NGS approach to study mutations induced by benzo[a]pyrene (BaP) in Muta™Mouse bone marrow.
- To improve the sensitivity and efficiency of the mutation reporter assay.
Main Methods:
- Developed a high-throughput NGS approach for simultaneous sequencing of barcoded mutant lacZ transgenes.
- Collected 3872 mutants from Muta™Mouse bone marrow exposed to benzo[a]pyrene (BaP) and controls.
- Pooled, barcoded, and sequenced mutants using NGS.
Main Results:
- Identified 1652 mutant sequences from 1006 independent mutations with clonal expansion.
- Confirmed BaP primarily causes guanine transversions (G:C → T:A), consistent with prior studies.
- Discovered novel mutational hotspots in the lacZ transgene and improved assay sensitivity by 50% by correcting for clonal expansion.
Conclusions:
- NGS offers a high-throughput, cost-effective, and sensitive method for studying mutagenic mechanisms.
- This approach facilitates comparison of mutagenicity across tissues and enhances genotoxin evaluation.
- The developed NGS method provides a fast and efficient tool for genotoxicity testing.
