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