Analysis of Cre-mediated excision of the reprogramming STEMCCA-loxP vector genomic insert from iPSCs and demonstration of continued expression of native pluripotency genes following excision. (A) Shown is the PCR (left) and sequencing (right) analyses of the residual genomic footprint left after Cre-mediated loxP excision from single-copy reprogramming STEMCCA-loxP vector genomic insert from one of the iPSC lines reprogrammed from nonmobilized CD34⁺ cells derived from peripheral blood. Following Cre-GFP transfection, 20% to 75% of all colonies from all donors tested had the vector removed and no colonies retained GFP expression. For this representative cell line, LAM-PCR analysis was used to determine the genomic site of the single-copy insert of STEMCCA-loxP. Primers were designed that matched human genomic sequences at least 300 to 500 bp flanking the insert and were used to generate standard PCR products from genomic DNA as shown in this agarose gel image on the left. The left lane in the gel is the 100-bp sizing ladder used to estimate the sizes of the PCR products in this example. The right lane shows that 2 bands approximately 300-bp different in size are generated from the primer pair, where the lower band is derived from the unaltered allele’s native genomic sequence of approximately 800 bp that separates the primer binding sites, while the upper band is approximately 300-bp larger derived from the allele that contains the same intervening human sequence plus the residual vector footprint following Cre-mediated loxP excision. The footprint sequence was determined by cloning and sequencing this upper band PCR product. Shown on the right is the entire 303-bp residual STEMCCA-loxP footprint sequence consisting of 1 complete vector LTR sequence (black) with the 5′ and 3′ portions of that LTR sequence separated by a single intervening loxP site derived from the recombination excision event. The 2 palindromic ends of the loxP are shown in red while the nonpalindromic middle section of the loxP sequence is shown in blue. (B) Bar graphs showing quantitative reverse-transcription PCR detection of expression of mRNAs encoding for pluripotent markers NANOG (red), OCT4 (green), and TERT (blue) in several representative CD34⁺ cell-derived iPSCs from which STEMCCA-loxP had been excised with Cre. Expression in the Cre-excised iPSC lines are compared with H9 ESCs with the TaqMan-based expression data normalized to expression of human B2M (β-2-microglobulin) as an endogenous control and expressed in relative units for comparison. PBMCs were used as the calibrator control for expression comparisons. All samples were analyzed in quadruplicate.