HBB and HBG transcription and translation analyses. (A) HBB and HBG1/2 gene expression (normalized to a house-keeping gene GAPDH) in undifferentiated iPSCs (S1) and differentiated progenies (EB and EryB) of S1, cre4, or cre16 was measured by quantitative RT-PCR (data represent mean ± SEM, n = 3). After the erythroid differentiation from iPSCs, the HBB transcript level increased 10- to 100-fold, although it is still 100- to 1000-fold lower compared with the level in CB-MNCs. (B) Conventional RT-PCR that readily amplifies HBB cDNA in erythroblasts derived from various SCD iPSC clones before (S1) or after gene targeting (c36) and Cre-mediated excision (cre4 and cre16), by 2 primers located at exon 1 and 3 (left illustration). (C) Although sizes of RT-PCR products of the unmodified or corrected alleles are the same, DNA sequencing of the RT-PCR product showed uniform transcript in c36-EryB and mixed transcripts in cre4-EryB and cre16-EryB (bottom chromatographs). Cloning each transcripts into TOPO vector and sequencing at clonal levels will distinguish expression from corrected versus uncorrected alleles. Sequencing of 40 to 60 individual cloned DNA molecules of RT-PCR products from each differentiated iPSC line revealed that the absence of corrected HBB transcript (T, 100% or 40/40 cloned and sequenced) in c36-derived erythroblasts, but in the erythroblasts derived from cre4 and 16 after Cre-mediated excision of the PGK-Hyg gene cassette, expression of the corrected (A) allele was detected. In cre4, both corrected (A, 28% or 17/60) and the unmodified (and mutated T) HBB alleles (72% or 43/60) were expressed. A similar result was obtained in cre16 iPSCs: 12/60 (20%) and 48/60 (80%) of the cloned and sequenced transcripts are from the corrected (A) and the uncorrected (T) alleles, respectively. (D) S1-EryB, cre4-EryB, and cre16-EryB expressed abundant fetal-type hemoglobin HbF, but no detectable adult-type hemoglobin HbA measured by flow cytometry using specific antibodies.