EVALUATION OF A PLASMID-BASED TRANSGENIC MOUSE MODEL FOR DETECTING IN-VIVO MUTATIONS

To study in vivo somatic mutations a C57BL/6 transgenic mouse model wa s constructed harboring multiple chromosomally integrated copies of th e plasmid pUR288, which carried the lacZ reporter gene as the mutation al target, We previously demonstrated that lacZ-containing plasmids co uld be rescued from their integrated state efficient enough to detect mutations in lacZ by positive selection, The smaller size of the plasm id vector, as compared with our earlier transgenic mouse model based o n bacteriophage lambda vectors, should offer considerable advantages i n terms of rescue efficiency and sensitivity to large size alterations in the lacZ gene, To evaluate the plasmid-based mouse model for its s uitability to detect in vivo mutations, we determined mutant frequenci es in different organs of untreated and ethyl nitrosourea (ENU)-treate d animals using a new, improved protocol, The rescue efficiencies obta ined were as high as 200 000/mu g genomic DNA; millions of transforman ts could be obtained in one single experiment, The average spontaneous mutant frequency in four different organs of 4- to 8-week-old mice ra nged from 4.41 to 6.82x10(-5), compared with a mutant frequency of the same plasmid grown in Escherichia coil of similar to 1x10(-5) or less , Single treatments with 100 and 250 mg ENU/kg body wt resulted in a 7 - and 14-fold increase, respectively, in spleen mutant frequency at 14 days after i.p. administration of the alkylating agent, Restriction e nzyme analysis showed that a considerable portion of spontaneous mutan ts were size changes varying from similar to 100 to 3000 bp, Some muta nt plasmids contained mouse genomic sequences, which is indicative of large genetic rearrangement events involving the 3' flanking regions o f the transgene cluster, Among the ENU-induced mutants, size changes c omprised only a minor fraction of the total, which is in keeping with the known ENU mutation spectra in vitro and in vivo. The high rescue e fficiency of this plasmid-based model, in combination with its sensiti vity to a broad spectrum of mutations, including large deletions, make s it very suitable as a general in vivo mutagenicity test system.