PhiC31 Integrase Mouse iPSC Reprogramming System

With both iPSC-generating donor and PhiC31 Integrase Expression plasmids, this kit delivers a complete, non-viral integrase-based iPSC generation system

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

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

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1 Kit
FC300A-1
$ 1482
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Overview

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.

pCOBLW (CAG-Oct4-Sox2-Klf4-Myc-GFP-SV40-Neo) Mouse Reprogramming PhiC31 Donor PlasmidPhiC31 Integrase Expression Plasmid

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.

How It Works

One-step transgene delivery with the PhiC31 Integrase System

With the PhiC31 Integrase System, you simply clone your gene-of-interest into the attB Donor Plasmid, and then (Step 1) co-transfect with a PhiC31 Integrase Expression Plasmid. (Inside the cell) The PhiC31 Integrase is transiently expressed, and mediates site-specific recombination between the attB site on the donor plasmid and a pseudo attP site in the genome. (Result) Because pseudo attP sites are typically present in transcriptionally active sites of the genome, your gene-of-interest will likely be integrated into an active region of the genome.

One-step transgene delivery with the PhiC31 Integrase System

Supporting Data

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

With the PhiC31 Integrase-based reprogramming system, the majority of clones that undergo integration events have single insertions

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.

The PhiC31 Integrase System efficiently generates mouse pluripotent stem cells

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 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.