The Gp1ba promoter drives high-level expression in iPSC-derived megakaryocytes. (A) Schematic of the targeting construct. The Gp1ba expression construct (gray window) contains the murine proximal Gp1ba promoter linked to the Simian virus 40 (SV40) 5′ untranslated region (UTR) followed by a cDNA of interest (eGFP shown) followed by the SV40 3′ UTR.¹⁰  After insertion into the puromycin (puro) containing targeting plasmid (pAAVS1-SA-2A-puro-pA), the entire transgene is inserted into the AAVS1 locus (PPP1R12C intron 1) using zinc finger nuclease–mediated homologous recombination. After insertion, the endogenous PPP1R12C promoter drives puromycin resistance (gray arrow), while the Gp1ba promoter drives the transgenic message (black arrow) (SA, splice acceptor; 2A, self-cleaving peptide; pA, bovine growth hormone polyadenylation signal). (B) Control 2 with hemizygous insertion of the Gp1ba promoter driving eGFP was examined by flow cytometry during hematopoietic differentiation. (Left) Representative plots of SSEA3⁺/SSEA4⁺ iPSCs, KDR⁺/CD31⁺ mesoderm, and CD41⁺/CD235⁺ HPCs. (Right) Histograms of gated populations on left (gray rectangles) examine eGFP expression in iPSCs, mesoderm, and HPCs. (C) (Left) Representative flow cytometry plots of iPSC-derived CD45⁺/CD18⁺ myeloid cells, CD41^–/CD235⁺ erythroid cells, and CD41⁺/CD42⁺ megakaryocytes. (Right) Histograms of gated populations on left (gray rectangles) examine eGFP expression in iPSCs, myeloid cells, erythroid cells, and megakaryocytes. (D) Mean fluorescence intensity (MFI) of eGFP expression in iPSCs, mesodermal cells (meso), HPCs, and megakaryocytes (megs). (E) Comparison of gene expression by quantitative reverse transcriptase polymerase chain reaction of endogenous GP1BA, GP1BB, and GP9 in iPSC-derived HPCs and megakaryocytes relative to cyclophilin (mean ± standard error of the mean [SEM] for 3 replicates).