BACKGROUND: DNA transposons have emerged as indispensible tools for manipulating 
vertebrate genomes with applications ranging from insertional mutagenesis and 
transgenesis to gene therapy. To fully explore the potential of two highly 
active DNA transposons, piggyBac and Tol2, as mammalian genetic tools, we have 
conducted a side-by-side comparison of the two transposon systems in the same 
setting to evaluate their advantages and disadvantages for use in gene therapy 
and gene discovery.
RESULTS: We have observed that (1) the Tol2 transposase (but not piggyBac) is 
highly sensitive to molecular engineering; (2) the piggyBac donor with only the 
40 bp 3'-and 67 bp 5'-terminal repeat domain is sufficient for effective 
transposition; and (3) a small amount of piggyBac transposases results in robust 
transposition suggesting the piggyBac transpospase is highly active. Performing 
genome-wide target profiling on data sets obtained by retrieving chromosomal 
targeting sequences from individual clones, we have identified several piggyBac 
and Tol2 hotspots and observed that (4) piggyBac and Tol2 display a clear 
difference in targeting preferences in the human genome. Finally, we have 
observed that (5) only sites with a particular sequence context can be targeted 
by either piggyBac or Tol2.
CONCLUSIONS: The non-overlapping targeting preference of piggyBac and Tol2 makes 
them complementary research tools for manipulating mammalian genomes. PiggyBac 
is the most promising transposon-based vector system for achieving site-specific 
targeting of therapeutic genes due to the flexibility of its transposase for 
being molecularly engineered. Insights from this study will provide a basis for 
engineering piggyBac transposases to achieve site-specific therapeutic gene 
targeting.