TALEN-Mediated Targeted Gene Integration: Using Engineered Sequence-Specific Nucleases for the Precise Insertion of Fluorescent Protein Gene into hiPSCs

Begin with human induced pluripotent stem cell or hiPSC suspension in media. Add a pair of transcription activator-like effector nuclease or TALEN-encoding plasmids. Supplement with a donor plasmid bearing a fluorescent protein gene, coupled to an antibiotic resistance gene, with adjacent homology arms for specific alignment to target loci.

Electroporate the mixture to generate transient pores in the cell membranes, enabling plasmid internalization. Once inside the cells, the TALEN-encoding plasmids get processed, producing TALENs - chimeric proteins composed of a site-specific TALE DNA-binding domain fused to a non-specific FokI restriction endonuclease cleavage domain.

Each TALEN monomer via its DNA-binding domain - comprising tandem amino acid repeat modules - recognizes and binds to target loci, one module per nucleotide, on each DNA strand in opposing orientations, separated by a spacer sequence. FokI domains then dimerize and cleave DNA within the spacer sequence, creating double-strand breaks with overhangs.

In the presence of donor plasmid containing homology arms complementary to regions flanking the cleaved site, homology-directed repair occurs utilizing the homologous sequence as template for DNA repair synthesis to bridge the double-strand breaks. Following ligation of the nicks, the intervening fluorescent protein and antibiotic-resistance genes get integrated into the host genome.

Treat the hiPSCs, seeded on a feeder cell layer to support growth, with antibiotic-containing media. The promoter-less antibiotic resistance gene - driven by an upstream endogenous promoter - facilitates selection of TALEN-edited cells.

Begin by washing the iPSC cultures one time each with PBS, then add 1 milliliter of 37 degrees Celsius gentle cell dissociation reagent to each well, and incubate the cells at 37 degrees Celsius for approximately 5 minutes. When greater than 50% of the cells have dissociated from the culture vessel, use a P1000 pipette to mechanically disrupt any remaining cell clumps or attached cells. Then, add 2 milliliters of E8 medium to each well, and use a 10-milliliter pipette to further disaggregate the cultures into single-cell suspensions.

Now, pour the harvested cells into a 15-milliliter conical tube and spin them down. Resuspend the pellet in a minimal amount of E8 medium for counting. Then, after confirming the viability of the cultures by trypan blue exclusion as well as their sufficient dissociation, transfer 3 million cells into each of two 15-milliliter conical tubes.

While the cells are centrifuging, set the electroporation system to the cell-type specific program for the human embryonic stem cell line, H9. Then, add 10 micrograms of the homologous recombination donor alone to 1 pellet for the control sample, and 10 micrograms of the homologous recombination donor plasmid along with 5 micrograms of each TALEN plasmid for the experimental sample.

Next, add 100 microliters of room temperature complete P3 primary cell transfection solution to each of the control and experimental pellets, and resuspend the cells. Transfer the samples into individual cuvettes, and then, electroporate the samples. Immediately after the transfection, add 500 microliters of room temperature E8 medium to each cuvette, and then, transfer the transected iPSC samples drop-wise into individual 10-centimeter dishes containing DR4 mouse embryonic fibroblasts.