Integrating and expressing stably a transgene into the cellular genome remain 
major challenges for gene-based therapies and for bioproduction purposes. While 
transposon vectors mediate efficient transgene integration, expression may be 
limited by epigenetic silencing, and persistent transposase expression may 
mediate multiple transposition cycles. Here, we evaluated the delivery of the 
piggyBac transposase messenger RNA combined with genetically insulated 
transposons to isolate the transgene from neighboring regulatory elements and 
stabilize expression. A comparison of piggyBac transposase expression from 
messenger RNA and DNA vectors was carried out in terms of expression levels, 
transposition efficiency, transgene expression and genotoxic effects, in order 
to calibrate and secure the transposition-based delivery system. Messenger RNA 
reduced the persistence of the transposase to a narrow window, thus decreasing 
side effects such as superfluous genomic DNA cleavage. Both the CTF/NF1 and the 
D4Z4 insulators were found to mediate more efficient expression from a few 
transposition events. We conclude that the use of engineered piggyBac 
transposase mRNA and insulated transposons offer promising ways of improving the 
quality of the integration process and sustaining the expression of transposon 
vectors.