Evaluating TALEN-mediated DNA Repair in HeLa Cells

TALEN expression constructs (TAL 256 and TAL 278) were previously constructed with the Joung Lab REAL Assembly TALEN kit (Addgene, Watertown, MA, USA) [25 (link),61 (link)]. To assess DNA repair, HeLa cells (0.6 million) were seeded a day before transient transfection, co-transfected with a mCherry/GFP reporter plasmid and pairs of TALEN-expressing constructs [TAL 256 (200 ng) and TAL 278 (200 ng)] at a ratio of 1:3 [DNA (μg): Transfection reagent (μL)] with Viafect Transfection reagent (Promega, Madison, WI, USA). As a control, the reporter plasmid was co-transfected with empty vector [JDS70 (200 ng) and JDS 78 (200 ng)]. Medium was changed after 4 h incubation and replaced with complete medium with or without mirin (Sigma Aldrich, St. Louis, MO, USA; 100 μM). After 48 h, cells were tested for mCherry and GFP fluorescence by fluorescence microscopy and flow cytometry.