How to use piggyBac system in vitro and in vivo?
Our piggyBac system contains two vectors, both engineered as E. coli plasmids. One vector, referred to as the “helper plasmid”, encodes the transposase. The other vector, referred to as the “transposon plasmid”, contains two inverted terminal repeats (ITRs) bracketing the region to be transposed. The gene (or other DNA fragment) to be delivered into host cells is cloned into this region between the ITRs.
When both the helper and transposon plasmids are co-transfected into target cells, the transposase produced from the helper recognizes the two ITRs on the transposon, and inserts the flanked region (including the two ITRs) into the host genome. Insertion typically occurs at host chromosomal sites that contain the TTAA sequence, which is duplicated on the two flanks of the integrated fragment.
In cell culture systems, using a piggyBac vector system is relatively straightforward, with serial transfection or co-transfection of the transposon and helper plasmids. Alternatively, electroporation may be used instead of transfection.
In live animals, however, plasmid delivery is more problematic, with low transfection and electroporation efficiencies making co-delivery of two plasmids inefficient. Some researchers have made use of transgenic animal lines expressing either the transposon or transposase alone. Crossing of these two lines or transfection of a single plasmid into one of these transgenic lines can induce transposition of the transposon in live animals (e.g. Ding et al. Cell. 122:473-83 and Horn et el. Genetics. 163:647-61)