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. Author manuscript; available in PMC: 2015 Dec 1.
Published in final edited form as: Nat Biotechnol. 2015 Mar 23;33(6):646–655. doi: 10.1038/nbt.3178

Figure 4. Engineering chr7q deletions in normal hPSCs.

Figure 4

(a) Overview of strategy for engineering chromosome 7q deletions. An AAV carrying a positive (puro) and a negative (HSV-tk) selection gene is targeted into chr7q and correctly targeted clones are positively selected in a first step. In a second step, Cre recombinase is transiently expressed and clones that have lost the targeted copy of chr7q are selected with GCV. IDLV, integrase-deficient lentiviral vector.

(b) aCGH specific for chromosome 7 in all engineered del(7q)-hPSC clones analyzed, as indicated. The blue color indicates deletion (1 copy), the red color amplification (3 copies) and the white color normal diploid dosage. Lower panel: chromosome 7 ideogram. The purple box indicates the ~40 Mb region (approximately 112,920,418 – 152,127,281) functionally mapped in the panel of clones with engineered chromosome 7q deletions. Its 5′ and 3′ borders are defined by the 3′ border of the deletion in clone N-2.12.D-6Cre6 and the 3′ end of the deletion in clone H1-D-2Cre6, respectively.

(c, d) CD45 expression at day 14 of hematopoietic differentiation of del(7q)- engineered clones derived from the H1 hESC (c, upper panel and d, left panel) and the N-2.12 iPSC (c, middle and lower panels and d, right panel) line. Mean and SEM are shown. *P < 0.05, **P < 0.01, ***P < 0.001, n.s.: not significant.

(e) Methylcellulose assays at day 14 of hematopoietic differentiation. The number of colonies from 5,000 seeded cells is shown. (CFU-GEMM: colony-forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte; CFU-GM: colony-forming unit-granulocyte, monocyte; CFU-G: colony-forming unit-granulocyte; CFU-M: colony-forming unit-monocyte; BFU-E: burst-forming unit-erythrocyte)

(f) Schematic summary of the approach to defining the chr7q critical region. The purple box indicates the ~40 Mb region functionally mapped in the panel of clones with engineered chromosome 7q deletions from b. The orange box denotes the ~30 Mb region duplicated in the rescued clone MDS-2.A3C, shown in Fig. 3h. Their overlap defines a critical region of ~20 Mb spanning cytobands q32.3-q36.1 (nucleotides approximately 131,706,336- 152,127,281).