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. 2021 Mar 17;108(4):709–721. doi: 10.1016/j.ajhg.2021.03.005

Figure 3.

Figure 3

ERF depletion elevates γ-globin expression in vivo

(A) The experimental design for in vivo functional validations of ERF. ERF-targeted gRNA and Cas9 protein were electroporated into CD34+ HSPCs and after 24 h engrafted into immunodeficient mice by intravenous tail injection. Bone marrow (BM) and peripheral blood (PB) cells were harvested at week 16 after engraftment for further analysis.

(B) Flow cytometry analysis in mouse BM 16 weeks after transplantation for determination of human engraftment rates (left), human cell multilineage reconstitution (myeloid and B cells, middle), and human erythroid cells (right).

(C) Determination of the indel frequencies by Synthego analysis after sequencing of PCR products in PB and BM from engrafted mice.

(D) Measurement of HBG mRNA expression by quantitative real-time PCR in mouse BM 16 weeks after engraftment (n = 4 for edited mice and n = 7 for unedited mouse controls). Data are presented as means ± SD (∗∗p < 0.01; ∗∗∗p < 0.001).