Single or Multiple integrations with a single transfection with Re-excision capability

The Super PiggyBac transposase transient expression vector and PB513B-1 were co-transfected into HeLa cells and puromycin selection applied for 10 days (10ug/ml), (a.). Cells efficiently transposed were Puro resistant and GFP positive. In (b.), Human 293 cells were transfected with the Super PiggyBac transposase transient expression vector and PB531A-1. The cells were photographed after 7 days, virtually all cells were RFP positive.

Integrate multiple PB vectors simultaneously - Triple Integration

Three different PiggyBac transposon vectors (PB513B-1+PB533A-1+PB531A-1) were co-transfected with or without the Super PiggyBac transposase expression vector (PB200PA-1) into Human HT1080 cells. Puromycin selection (2ug/ml) and Neomycin (2mg/ml) were applied for 7 days. The +PiggyBac transposase cells were Puro and Neo resistant, GFP positive and RFP positive. Background GFP positive cells that are Puro resistant on the right side are due to random PB513B-1 integrations during the puromycin selection. The non-PiggyBac mediated integration rate in those cells was extremely low and no RFP positive cells were identified. It's easy and efficient to integrate multiple genes simultaneously using multiplexed transfections in conjunction with the Super PiggyBac transposase.

PiggyBac Transposon Re-excision

The integrated PiggyBac transposons can be successfully removed, footprint-free. Simply re-transfect your stably transposed cell line with increasing amounts of the Super PiggyBac transposase expression vector to mobilize and excise the integrated transposons. Shown below is an example of excising PB531A-1 RFP transposons from HEK293 cells using increasing amounts of re-transfected Super PiggyBac plasmid DNA (PB200PA-1). We achieved greater than 88% removal of the integrated RFP transposon using 2.5ug of re-transfected Super PiggyBac transposase expression vector plasmid.