Characterisation of Muta™Mouse λgt10-lacZ transgene: evidence for in vivo rearrangements.
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 CD2F(1) 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.
Shwed
Philip S
PS
Mechanistic Studies Division, Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario K1A 0K9, Canada. phil_shwed@hc-sc.gc.ca
Crosthwait
Jennifer
J
Douglas
George R
GR
Seligy
Vern L
VL
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D005260:Female; D007763:Lac Operon / Q000235:genetics*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008811:Mice, Inbred DBA; D008822:Mice, Transgenic / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology; D009152:Mutagenicity Tests / Q000379:methods; D011995:Recombination, Genetic / Q000235:genetics*; D017422:Sequence Analysis, DNA; D019076:Transgenes / Q000235:genetics*