Creation and characterization of IK6-expressing B-ALL via knock-in of IK6 into the endogenous IKZF1 locus. (A) HDR strategy for GFP-fused IK6 knock-in using 3.2 kb double-stranded DNA HDR template. Targeted exon 3 shown in purple; red indicates noncoding exon 1. *Mutated protospacer-adjacent motif (PAM) to prevent cleavage of template; vertical black rectangles denote zinc fingers. (B) Flow cytometry plots showing GFP⁺ sort gate used to purify cells that had IK6-GFP knock-in. (C) IK6-GFP fusion protein in nucleus and mislocalization to cytoplasm as shown by live cell confocal microscopy. Upper row, Nalm-6 GFP-positive pool showing heterogeneous expression 1 month after sorting; lower row, Reh single cell–derived clone. All images were taken on the GE Healthcare DeltaVision LIVE High Resolution Deconvolution Microscope using an Olympus 40X U Apo/1.35 NA oil objective with iris and analyzed with SoftWoRx. Scale bars are 20 µm. (D) Immunoblotting for IKAROS and GFP-IK6 fusion protein in Nalm-6 and Reh single cell–derived clones. (E) In vitro competition of Nalm-6 (upper graph) and Reh (lower graph). GFP was measured by flow cytometry at indicated time points. All samples were performed in triplicate, and error bars show standard error of the mean. Additional details are provided in supplemental Figure 5C-D.