Non-Viral PhiC31 Integrase Mouse iPSC Reprogramming System (includes FC200PA-1 and FC305A-1)

One-step, non-viral, generation of induced pluripotent stem cells (iPSCs)

Product Description

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

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 Integrase Expression Plasmid using GFP fluorescence and select for positive colonies using the built-in neomycin resistance marker.

How it works

One-step transgene delivery with the PhiC31 Integrase System

Supporting Data

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 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.