citations

                    Transcription activator-like effector nuclease (TALEN)-mediated CLYBL targeting enables enhanced transgene expression and one-step generation of dual reporter human induced pluripotent stem cell (iPSC) and neural stem cell (NSC) lines.
                

Authors:

Cerbini T, Funahashi R, Luo Y, Liu C, Park K, Rao M, Malik N, Zou J.

In:

Source: PLoS ONE
Publication Date: (2015)
Issue: 10(1): 1-18

Research Area:

Cardiovascular

Neurobiology

Stem Cells

Gene Expression

Cells used in publication:

Neural stem cell (NSC), human: Species: human; Tissue Origin: brain
Induced Pluripotent Stem Cell (iPS), human: Species: human; Tissue Origin:

Platform:

4D-Nucleofector® X-Unit

Experiment

a) 3x10e6 human iPSC (80% confluent before harvested with Accutase); P3 solution, 5µg TALEN plasmid and 10µg donor plasmid, program CB-150; cells were plated on MEF feeder cells in the presence of ROCK inhibitor for 24h; b) 3.5 x10e6 human NSCs, harvested with Accutase, P4 solution, 7µg DNA in total, program DN-100, DNA: TALEN right, TALEN left, donor vector: 1:1:1 After nucleofection cells were plated on geltrex coated 6 well plates.

Abstract

Targeted genome engineering to robustly express transgenes is an essential methodology for stem cell-based research and therapy. Although designer nucleases have been used to drastically enhance gene editing efficiency, targeted addition and stable expression of transgenes to date is limited at single gene/locus and mostly PPP1R12C/AAVS1 in human stem cells. Here we constructed transcription activator-like effector nucleases (TALENs) targeting the safe-harbor like gene CLYBL to mediate reporter gene integration at 38%-58% efficiency, and used both AAVS1-TALENs and CLYBL-TALENs to simultaneously knock-in multiple reporter genes at dual safe-harbor loci in human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs). The CLYBL-TALEN engineered cell lines maintained robust reporter expression during self-renewal and differentiation, and revealed that CLYBL targeting resulted in stronger transgene expression and less perturbation on local gene expression than PPP1R12C/AAVS1. TALEN-mediated CLYBL engineering provides improved transgene expression and options for multiple genetic modification in human stem cells

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