Mutation burst by conflict of two mouse subspecies genomes

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We carried out two experiments to elucidate mechanism by which high-frequency spontaneous mutations(Recombination-induced-mutation : Rim) is induced in mice with recombinant MHC haplotypes between two mouse subspecies. First, we tried to detect genome alterations similar to Rim by PCR-based monitoring of Rim-type mutations using pooled sperm DNAs of intra-MHC recombinant mice. As a result, we could not observe expected PCR-products by this method. One possibility to explain this result is that there are different types of mechanism for induction of Rim. Alternatively, present intra-MHC recombinant mice have lost the activity to give rise to Rim. Second, we tried to develop an efficient assay system to detect DNA-double strand breaks and subsequent recombination for single male mouse. To do this, now we constructed the DNA construct that consists of recombination-target DNA sequence for Prdm9 protein, which is known to determine recombination site by introducing Histon3 chemical modification as hotspot mark, and of GFP reporter connected to the Prdm9 target sequence. Now we are introducing this construct into mouse genome by means of Tol2 transposon system.