Mutagenesis in mice and rats using mobile DNA

Presented by:

Dr. David Largaespada, Professor, University of Minnesota

Agenda

Transposons are being used as tools to further initiatives to create comprehensive resources of mutant mice and rats strains. T he Sleeping Beauty (SB) transposon system is one such tool. Other transposable element systems also function in mice and rats. Novel germline insertion mutants can be generated by mobilizing transposon vectors from microinjected plasmids into chromosomes of single cell embryos, or by mobilizing transposon vectors from donor loci in developing germ cells of transgenic mice. Due to its tendency for local hopping, SB has been proposed as a method for regional saturation mutagenesis of the mouse genome. We have investigated SB as a tool for regional, saturation insertional mutagenesis in a forward-genetic screen for recessive lethal and viable mutants on distal mouse chromosome 11. We recovered visible and behavioral mutants, as well as a high frequency of recessive lethal mutations. Surprisingly, we discovered that most of the lethal mutations belonged to one of two complementation groups and arose as a result of local genomic rearrangements caused by transposon mobilization. These chromosomal rearrangements include inversions, deletions, and insertions of large segments of the donor concatemer and adjacent DNA into another chromosome. We have observed similar results with multiple donor concatemers and also during somatic cell transposition. Our results show that these types of genetic lesions often occur in conjunction with local transposition events in chromosomes. Our data indicate that while SB mobilization in the germline can generate transposon insertion-mediated phenotypes, additional phenotypes can result from associated genomic rearrangements. We conclude that local saturation mutagenesis of the germline will require measures to eliminate recurrent chromosomal deletions from such a screen. Alternatively, genome-wide, transposon-mediated forward genetic screens might be possible, but are only going to be practical after improving the germline transposition rate. In contrast, our data indicates that the SB system is ideally suited to forward genetic screens for cancer genes. The system requires creating mice that harbor both a transposon array of the insertionally mutagenic SB vector, T2/Onc, and express the transposase enzyme in the target somatic tissue. If transposition can induce cancer, then tumor DNA is studied by cloning insertion sites. These insertion sites are analyzed and we look for T2/Onc insertions at reproducibly mutated genes. Results from ubiquitous and tissue-specific transposon mutagenesis for cancer gene discovery will be presented.

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