PiggyBac Transposon-Mediated Gene Editing in Human iPSCs: A Procedure to Integrate Gene of Interest in Human iPSCs Using PiggyBac Transposon System

Begin with a suspension of human-induced pluripotent stem cells, hiPSCs, in suitable electroporation buffer. Add a solution consisting of PiggyBac transposase-encoding plasmids and donor plasmids. Electroporate the mixture.

The electrical pulses transiently permeabilize cell membranes, facilitating cellular uptake of plasmids. The donor plasmids comprise transposon-specific inverted terminal repeats, ITRs, that are inverted complements of each other, flanked by TTAA repeats bracketing the target protein and antibiotic resistance genes.

Expressed PiggyBac transposase proteins create nicks at the 3' ends of the ITRs, internal to the TTAA repeats. The exposed 3'-hydroxyl groups attack the complementary strand's nucleotide inside the flanking DNA, forming TTAA hairpins on the transposon ends, thereby releasing transposon construct from the plasmid.

Transposases resolve the hairpins to form TTAA overhangs at the 5' ends of the transposon construct. Transposases further create double-strand breaks at the TTAA sequence in the host genome. The released 3'-hydroxyl transposon ends attack the TTAA sequence at the staggered ends, resulting in covalent joining of the transposon construct to the host genome, leaving single-strand gaps flanking the transposon construct. These gaps in the host DNA are ligated, leading to stable integration of the transposon construct.

Treat the hiPSCs seeded on an extracellular matrix protein-coated plate with antibiotic-containing media. The antibiotic resistance gene, driven by the upstream promoter, enables selection of gene-edited cells.

Rinse the human iPSC culture with calcium and magnesium-free PBS, before treating the cells with cell dissociation reagent for 5 to 10 minutes at 37 degrees Celsius. When the cells have begun to detach from the culture container, use a P1000 pipette to gently manually dissociate the cells 3 to 4 times.

Transfer the cells to a 15-milliliter tube, and bring the final volume up to 10 milliliters with fresh PBS without calcium and magnesium, for counting. Collect 1 x 10⁶ cells by centrifugation, and re-suspend the pellet in 100 microliters of buffer R from the electroporation kit.

Add 4.5 micrograms of transposable vector plasmid DNA, and 0.5 micrograms of the PiggyBac transposase plasmid DNA for transfection. Transfect with the cell electroporation system according to the manufacturer's instructions, with the indicated parameters. Then. seed the cells in human iPSC medium supplemented with 10 micromolar ROCK inhibitor Y-27632 in a 6-millimeter matrix-coated dish.