Nuclease-mediated double-strand break (DSB) enhancement of small fragment homologous recombination (SFHR) gene modification in human-induced pluripotent stem cells (hiPSCs).

Sargent RG, Suzuki S, Gruenert DC.
Source: Methods Mol Biol
Publication Date: (2014)
Issue: 1114: 279-90
Research Area:
Stem Cells
Gene Expression
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
4D-Nucleofector® X-Unit

8x10e5 cystic fibrosis derived iPS cells (detached with accutase) were transfected with small fragments DNA and TALEN plasmids in 100µl of P3 solution with program CB-150; 2 samples were pooled and divided afterwards in two wells of 24 well plate. - cells were cultured feeder free and were treated pre-and post nucleofection with ROCK Inhibitor


Recent developments in methods to specifically modify genomic DNA using sequence-specific endonucleases and donor DNA have opened the door to a new therapeutic paradigm for cell and gene therapy of inherited diseases. Sequence-specific endonucleases, in particular transcription activator-like (TAL) effector nucleases (TALENs), have been coupled with polynucleotide small/short DNA fragments (SDFs) to correct the most common mutation in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, a 3-base-pair deletion at codon 508 (delF508), in induced pluripotent stem (iPS) cells. The studies presented here describe the generation of candidate TALENs and their co-transfection with wild-type (wt) CFTR-SDFs into CF-iPS cells homozygous for the delF508 mutation. Using an allele-specific PCR (AS-PCR)-based cyclic enrichment protocol, clonal populations of corrected CF-iPS cells were isolated and expanded.