Elucidating the endogenous elements that regulate the CYBB gene. (A) Visual representation of the University of California, Santa Cruz Genome Browser displaying putative enhancers regulating the native CYBB gene. The CYBB gene is highlighted in the red box. Green arches represent the putative genomic sites interacting with the CYBB promoter. (B) A series of LVs were designed, each containing a single putative enhancer element cloned upstream of the parental construct. The CCLchim vector is used as a comparator of expression and regulation. Mean fluorescence intensity (MFI) of the mCitrine-positive population is shown for the human cord blood CD34⁺ differentiated neutrophils (C) and monocytes (D) as well as the RAMOs (E) and Jurkat cell lines (F). Data are presented as mean ± standard deviation (SD). Statistical significance was analyzed using an unpaired t test. All statistical tests were 2-tailed and P < .05 was deemed significant; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. Int3 enh, CYBB intron 3 enhancer; mCit, mCitrine; ns, nonsignificant; pro, CYBB promoter; tRNA, transfer RNA; WPRE, Woodchuck hepatitis virus posttranscriptional regulatory element.