Extended Data Fig. 3: Gene targeting approaches to create UMPSKO/KO cells and to knock-in Luciferase-GFP into a safe-harbor locus. | Nature Biotechnology

Extended Data Fig. 3: Gene targeting approaches to create UMPSKO/KO cells and to knock-in Luciferase-GFP into a safe-harbor locus.

From: Metabolic engineering generates a transgene-free safety switch for cell therapy

Extended Data Fig. 3

a, Gene targeting approach used to create UMPS-/- Nalm6 cells and UMPS-/- T cells. Dual-allelic gene targeting using constructs carrying tNGFR and tEGFR allow for the purification of the dual-positive population by either fluorescence-activated or magnetic beads-activated cell sorting (FACS or MACS). b, Representative FACS plots of Nalm6 cells that have undergone dual-allelic gene targeting at the UMPS locus and after sorting of the NGFR+/EGFR+ population. Controls are shown that were only mock electroporated or AAV transduced without RNP electroporation. Data from a single experiment. c, Approach used to create cells expressing Luciferase and GFP from a safe harbor. The depicted donor construct was used to target the HBB gene locus considered a safe harbor in non-erythropoietic cells. d, FACS analysis of K562 cells targeted with the approach in c and the relevant controls, before and after sorting of GFP+ cells. The procedure was performed once.

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