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.
Wu
Sen
S
Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah 84112, USA.
Ying
Guoxin
G
Wu
Qiang
Q
Capecchi
Mario R
MR
D000818:Animals; D015820:Cadherins / Q000235:genetics; D005260:Female; D016678:Genome; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011995:Recombination, Genetic; D014178:Translocation, Genetic
D015820:Cadherins