Nucleofection of U2OS derived cells with 500 ng of each TALEN plasmid DNA and 50 ng ptdTomato-N1 plasmid DNA using a Lonza 4D-Nucleofector System, Solution SE and program DN-100. As a negative control, 1 µg of ptdTomato-N1 plasmid was transfected alone. Cells were assayed for EGFP and tdTomato expression at 2 and 5 days post-transfection using flow cytometry.
The authors developed a system for large scale construction of transcription activator–like effector nucleases (TALENs) genome editing tools. The method, named fast ligation-based automatable solid-phase high-throughput (FLASH), is publically available, rapid and cost-effective. 1µg of TALEN plasmid pair (500ng each) were transfected into U2OS derived cells, using the 4D-Nucleofector™ System. Using this method, the authors were able to efficiently introduce targeted alterations in 84 out of 96 endogenous human genes implicated in cancer and epigenetic regulation.