Encyclopedia of Experiments: Biological Techniques
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Transcript
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.
