pCOBLW [CAG-Oct4-Sox2-Klf4-Myc-GFP-SV40-Neo] Mouse Reprogramming PhiC31 Donor Plasmid

PhiC31 Integrase-based reprogramming—non-viral induction of pluripotent stem cells

Figure 1. With the PhiC31 Integrase-based reprogramming system, the majority of clones that undergo integration events have single insertions. Southern blot analysis of genomic DNA from positive clones co-transfected with both the PhiC31 Integrase Expression Plasmid (Cat.# FC200PA-1) and the pCOBLW (CAG-Oct4-Sox2-Klf4-Myc-GFP-SV40-Neo) Mouse Reprogramming PhiC31 Donor Plasmid shows the copy number status for 16 different iPSC lines. With both restriction enzymes used, 14 out of 16 lines show single insertions (88%) demonstrating the tight copy number control possible with the PhiC31 Integrase System.

Figure 2. The PhiC31 Integrase System efficiently generates mouse pluripotent stem cells. Mouse iPSCs were generated by nucleofecting genetically unmodified C57BL/6 mouse embryonic fibroblasts with the pCOBLW (CAG-Oct4-Sox2-Klf4-Myc-GFP-SV40-Neo) Mouse Reprogramming PhiC31 Donor Plasmid (Cat# FC305A-1) and the PhiC31 Integrase Expression Plasmid (Cat.# FC200PA-1). iPSC cells were derived using morphological selection criteria and GFP expression without drug selection. When cultured under standard mouse ES cell culture conditions, the morphology of SBI mouse iPSCs are identical to that of mouse ES cells. The cells also express the pluripotency markers SSEA-1, Nanog, Sox2, and Oct4 and demonstrate strong endogenous AP activity.