Characterisation of Muta™Mouse ?gt10-lacZ transgene: evidence for in vivo rearrangements.

ABSTRACT: The multicopy ?gt10-lacZ transgene shuttle vector of Muta™Mouse serves as an important tool for genotoxicity studies. Here, we describe a model for ?gt10-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 ?gt10 used to construct the transgene and the Muta™Mouse precursor (strain 40.6), we validated the sequence integrity of key ? 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 CD2F(1) genome with variable ?gt10-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 ?gt10-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.

SUBMITTER: Shwed PS 

PROVIDER: S-EPMC2966988 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature