Description: Pronuclear microinjection of bacterial artificial chromosomes (BACs)is the preferred way to generate transgenic mice because the transgene accurately recapitulates expression of the endogenous gene. However, the method is demanding and the integrity and copy number of the BAC transgene is difficult to control. Here we describe a simpler pronuclear injection method that relies on transpositionto introducefull-length BACs into the mouse genome. The bacterial backbone of a hPAX6-GFP reporter BAC was retrofitted with PiggyBac transposon inverted repeats(ITRs) andco-injectedwith PiggyBac transposase mRNA. Both the frequency of transgenic founders as well as intact, full-length, single copy integrations was increased.Transposition was determined by a rapid PCR screen for a transpositional signature and confirmation by splinkerette sequencing to show that the BACs were integrated as a single copy either in one or two different genomic sites. BAC transposons displayed improved functional accuracy over random integrants as evaluated by expression of the hPAX6-GFP reporter in embryonic neural tube and absence of ectopic expression. This method involves less work to achieve increased frequencies of both transgenesis and single copy, full-length integrations. These advantages are not only relevant to rodents but also for transgenesis in all systems. © 2012 Wiley-Liss, Inc.