Generation of Molecularly Defined Inbred Rat Mutants

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DESCRIPTION (provided by applicant): The long-term goal of this project is to produce a cryopreserved repository of inbred rat strains each having a precisely defined loss of function mutation in a different known gene. Such a resource will be made available to all suitably qualified researchers in the field for minimal cost. This R21 application is based on a novel approach we have successfully developed in the mouse. It represents a proof-of-principle request in order for us to transfer the technology to the rat where it may be of even greater utility. We are confident that this approach will be successful in the rat and see this R21 funding as a stepping-stone to a larger project designed to create a community resource of mutant rats. Mutants will be produced in vivo using a unique coat color tagged, transposon mediated, gene trapping approach. An inbred transgenic albino rat (F344) carrying the Sleeping Beauty (SB) transposon vector, pT2/BART3 will be mated to an inbred F344 rat carrying a hyperactive SB transposase, driven by the proven male germ line specific promoter, PGK2: SB11. Male F1 rats carrying both transgenes will then be mated to normal albino F344 females. Any progeny that are pigmented will represent transposon jumps. The pigmentation occurs because the transposon vector, pT2/BART3 contains the tyrosinase minigene which is able to rescue albinism in rodents. When making the transgenic rats the vector is simply linearized for injection. The flanking vector sequences act to inhibit tyrosinase expression in the initial transgenic. If the transposon is mobilized to jump in the male germ line by the transposase, it is released from these inhibitory sequences, and the progeny becomes pigmented. As the vector also contains splice acceptors in both orientations, gene trapping makes jumps highly mutagenic. In our mouse model the jumping frequency is currently 50-60%. The great power of this approach is that the insertion points of the transposon jumps can be simply amplified from pre-weaning tail snip DNA, using degenerate oligo PCR, and sequenced. The precise integration point can then be identified by comparison with the recently available public rat genome sequence. Rats with gene trap transposon jumps into known or predicted genes, or other areas of interest, in the genome can be expanded for study or cryopreserved for distribution.