Zinc Finger Nuclease-Based Genome Editing: A Technique for Modifying Genome in Human Pluripotent Stem Cells by Double-Stranded Homology Dependant Repair Mechanism

Abstract

Source: Blair, J. D., et al. Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation. J. Vis. Exp. (2016).

This video demonstrates a technique of zinc-finger nuclease-based genome editing in human pluripotent stem cells (hPSCs). As hPSCs can potentially be differentiated into various cell types, this technology is helpful for in vitro pathological and pharmacological studies in a patient-specific manner.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Prepare Stem Cells for Editing

1. Grow and culture human pluripotent stem cells (hPSCs) on a 6-well plate containing 2.4 × 10⁶ cells/plate of mitomycin C-inactivated mouse embryonic fibroblast (MEF) feeders grown on gelatin. Maintain hPSCs in 3 ml of human embryonic stem cell media per well (hESC media) and grow in a 37 °C incubator with 3% O₂/5% CO₂.
   NOTE: For the success of this protocol, it is not nec....

Results

Figure 1. Schematic of gene edited AAVS1 locus using the AAV-CAGGS-EGFP repair template. Modified from Hockemeyer et al., 2009.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM/F12	Life Technologies	11320082
Fetal Bovine Serum (HI)	Life Technologies	10082-147
Knockout Serum	Life Technologies	10828-028
Fibroblast Growth Factor - basic	Life Technologies	PHG0261
Pen/Strep	Life Technologies	15140-122
Glutamine	Life Technologies	25030-081
MEM NEAA	Life Technologies	11140-050
2-mercaptoethanol	Life Technologies	21985-023
Y-27632	Calbiochem	688000
6-well plates	Corning	3506
4 mm Electroporation cuvettes	Bio-rad	165-2081
X-cel gene pulser II	Bio-rad	165-2661
0.25% Trypsin-EDTA	Life Technologies	25200-056
10× Phosphate buffered saline (PBS) pH7.4	Life Technologies	70011-044
Puromycin	Life Technologies	A11138-02