Characterisation of Muta™Mouse λgt10-lacZ transgene: evidence for in vivo rearrangements

• Published in: Mutagenesis 2010-11, Vol.25 (6), p.609-616
• Main Authors: Shwed, Philip S., Crosthwait, Jennifer, Douglas, George R., Seligy, Vern L.
• Format: Article
• Language: English
• Subjects: Animals; Bacteriophage lambda - genetics; Base Sequence; Biological and medical sciences; Cloning, Molecular; Escherichia coli; Female; Fundamental and applied biological sciences. Psychology; Lac Operon - genetics; Male; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Mice, Transgenic - genetics; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutagenesis - physiology; Mutagenesis. Repair; Mutagenicity Tests - methods; Original; Recombination, Genetic - genetics; Sequence Analysis, DNA; Transgenes - genetics
• ISSN: 0267-8357; 1464-3804
• DOI: 10.1093/mutage/geq048

The multicopy λgt10-lacZ transgene shuttle vector of Muta™Mouse serves as an important tool for genotoxicity studies. Here, we describe a model for λgt10-lacZ transgene molecular structure, based on characterisation of transgenes recovered from animals of our intramural breeding colony. Unique nucleotide sequences of the 47 513 bp monomer are reported with GenBank® assigned accession numbers. Besides defining ancestral mutations of the λgt10 used to construct the transgene and the Muta™Mouse precursor (strain 40.6), we validated the sequence integrity of key λ genes needed for the Escherichia coli host-based mutation reporting assay. Using three polymerase chain reaction (PCR)-based chromosome scanning and cloning strategies, we found five distinct in vivo transgene rearrangements, which were common to both sexes, and involved copy fusions generating ∼10 defective copies per haplotype. The transgene haplotype was estimated by Southern hybridisation and real-time–polymerase chain reaction, which yielded 29.0 ± 4.0 copies based on spleen DNA of Muta™Mouse, and a reconstructed CD2F1 genome with variable λgt10-lacZ copies. Similar analysis of commercially prepared spleen DNA from Big Blue® mouse yielded a haplotype of 23.5 ± 3.1 copies. The latter DNA is used in calibrating a commercial in vitro packaging kit for E.coli host-based mutation assays of both transgenic systems. The model for λgt10-lacZ transgene organisation, and the PCR-based methods for assessing copy number, integrity and rearrangements, potentially extends the use of Muta™Mouse construct for direct, genomic-type assays that detect the effects of clastogens and aneugens, without depending on an E.coli host, for reporting effects.