One-step, non-viral, generation of induced pluripotent stem cells (iPSCs)
Get the both of the plasmids you need—the PhiC31 Integrase Expression Plasmid (Cat.# FC200PA-1) and the pCOBLW (CAG-Oct4-Sox2-Klf4-Myc-GFP-SV40-Neo) Mouse Reprogramming PhiC31 Donor Plasmid—for SBI’s integrase-based reprogramming of murine cells.
The pCOBLW (CAG-Oct4-Sox2-Klf4-Myc-GFP-SV40-Neo) Mouse Reprogramming PhiC31 Donor Plasmid contains the coding regions of the murine Oct4, Sox2, Klf4, and cMyc genes, which are needed for generation of mouse iPSCs, and the PhiC31 Integrase Expression Plasmid provides the PhiC31 Integrase enzyme needed to catalyze integration of pCOBLW into the genome. Because the PhiC31 Integrase system delivers tight control over copy number, the majority of clones that undergo integration events have single insertions (Supporting Data, Figure 1), reducing the risks of insertional mutagenesis effects.
Popularly used for gene therapy development applications, the pCOBLW Reprogramming PhiC31 Donor Plasmid features the iPSC factors co-expressed by a potent CAG promoter. Co-expression is coordinated by the P2A elements at the 3’ end of each iPSC factor, including before the EGFP reporter gene. HS4 insulators flank the expression cassette and a functional attB site directs the integration of the entire plasmid into pseudo attP sites in any genome. The vector is engineered with FRT sites for later excision using standard FLP recombinase methods.
You can monitor co-transfection of the reprogramming vector with the PhiC31 Expression Plasmid (Cat.# FC200PA-1) using GFP fluorescence and select for positive colonies using the built-in neomycin resistance marker.
The PhiC31 Integrase System
SBI’s PhiC31 Integrase System consists of the PhiC31 Integrase Expression Plasmid and a PhiC31 Donor Plasmid that has your gene-of-interest cloned in. The system enables:
- Non-viral transgene delivery
- Single-copy integration
- Preferential integration at active sites
- Delivery of inserts with no size constraints
- Easy generation of cell lines
When you want non-viral gene delivery, have a large insert, and/or need controlled, single-copy genomic integration, turn to the PhiC31 Integrase System.