Toward simpler and faster genome-wide mutagenesis in mice

Here we describe a practical Cre- loxP and piggyBac transposon - based mutagenesis strategy to systematically mutate coding sequences and/ or the vast noncoding regions of the mouse genome for large- scale functional genomic analysis. To illustrate this approach, we first created loxP- containing loss-of-function alleles in the protocadherin alpha, beta and gamma gene clusters ( Pcdha, Pcdhb and Pcdhg). Using these alleles, we show that, under proper guidance, Cre- loxP site- specific recombination can mediate efficient trans- allelic recombination in vivo, facilitating the generation of large germline deletions and duplications including deletions of Pcdha, and Pcdha to Pcdhb, simply by breeding ( that is, at frequencies of 5.5% - 21.6%). The same breeding method can also generate designed germline translocations between nonhomologous chromosomes at unexpected frequencies of greater than 1%. By incorporating a piggyBac transposon to insert and to distribute loxP sites randomly throughout the mouse genome, we present a simple but comprehensive method for generating genome-wide deletions and duplications, in addition to insertional loss-of-function and conditional rescue alleles, again simply by breeding.