A subscription to JoVE is required to view this content.

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

-- views • 2:08 min

Transcript

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.

article

02:18

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

Related Videos

1.1K Views

article

14:46

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

Related Videos

11.0K Views

article

09:51

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Related Videos

13.4K Views

article

07:42

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs)

Related Videos

8.2K Views

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2025 MyJoVE Corporation. All rights reserved