Figure 3

A stronger and more silencing-resistant construct for efficient selection of bi-allelic excised iPSC clones.

(A) Analysis of transgene silencing in the TRA-1–60⁺ population of CAG-eGFP-IRES-puro or PGK-eGFP-IRES-puro transduced and puromycin-selected hPi cells, 7 days after puromycin withdrawal. Difference between identical bulk populations cultivated with and without puromycin was calculated, giving the % of transgene silencing. Average lentiviral vector copy numbers (VCN) were determined by qRT-PCR. (B) Representative FACS plots from CAG- or PGK-transduced hPi cells, grown for 7 days with or without puromycin. FACS-controls are given in Figure S2A. (C) Schematic of the new selection/ counter-selection cassette: the PGK promoter in the AAT-PB-PGK-puroΔtk plasmid was replaced by CAG and an eGFP or DsRedEx cassette was added over a T2A joint before the puroΔtk for better monitoring of transgene expression, resulting in AAT-PB-CG2ApΔtk or AAT-PB-CG2ApΔtk, respectively. (D) FACS sort for DsRed⁺ single positive (framed in red), eGFP⁺ single positive (framed in green) and DsRed⁺, eGFP⁺ double positive (framed in blue) hPi cells after transfection of AAT-PB-CG2ApΔtk + AAT-PB-CD2ApΔtk + Cas9_D10A + AAT_g1 + AAT_g2 and puromycin selection. (E) PCR analysis for the untargeted SERPINA1-allele in 23 established clonal cell lines generated from double-positive sorted cells. Genomic DNA from HEK293-T and hPi parental cell lines served as positive controls, whereas bi-allelic targeted hPi #8 was used as negative control. (F) PCR analysis of piggyBac excised, FIAU-resistant and puromycin-sensitive clones generated from bi-allelic targeted clones for left and right flanking arm bands and untargeted or excised allele (uta). Presence or absence of full length 2.5 kb genomic fragment spanning over the genomic location of both flanking arms was analyzed using forward primer from left band PCR and reverse primer from right band PCR. hPi and hPi #8 served as controls. Data are represented as mean ± SD and statistical analysis was performed by two-tailed student’s t-test where **p < 0.01, ***p < 0.001, n = 4 biological replicates (= individual transductions).