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