Make Stable Cell Lines using EV Shuttles and piggyBac

The piggyBac (PB) transposon is a mobile genetic element that efficiently transposes between vectors and chromosomes via a "cut and paste" mechanism. During transposition, the Super PB transposase recognizes transposon-specific inverted terminal repeat sequences (ITRs) located on both ends of the transposon vector and moves the contents from the original sites and efficiently integrates them into TTAA chromosomal sites.

Mouse stable ES cell lines

The Super PB transposase expression plasmid (5 ug) and 5 ug of the PB713B-1 PB transposon vector encoding markers for GFP and a Puromycin resistance gene were loaded into Human EV shuttles and then added to Mouse embryonic stem cells in culture. ES cells were placed under 0.5 ug/ml puromycin selection for 3 days initially and then increased to 1 ug/ml puromycin for an additional 4 days of selection. As control, EV shuttles were loaded without the Super PB transposase. The images below show the successful genomic integration of Human EV-shuttle mediated piggyBac transposons into mouse ES cells.

How do EV shuttles compare to Lipofectamine®?

The Super PB transposase expression plasmid (5 ug) and 5 ug of the PB713B-1 PB transposon vector encoding markers for GFP and a Puromycin resistance gene were used to compare the efficiency of transposition using either standard Lipofectamine methods or HEK293 EV shuttles. After 72 h post EV shuttle treatment, puromycin selection was initiated at 2 ug/ml and then increased to 5 ug/ml for 3 days further. EV shuttle-mediated transpositioned HEK293 cells were imaged post selection (upper panel) and compared to cells that were Lipofectamine transfected with the same PB plasmids (lower panel). Delivery of piggyBac vectors by EV shuttles was less toxic than transfection, highly efficient at PB transposon host chromosome integration, and resulted in approximately 5-times more colonies.