Today, I’d like to invite you to take a look at a highly efficient and useful kit, which brings together all the required components you need in a complete system for culturing and transfecting human pluripotent stem cells for gene editing.

The PluriQ™ G9™ Gene Editing System includes the G9™ Maintenance Medium and G9™ VTN Recombinant (vitronectin) plate coating for culturing human induced pluripotent (iPS) or embryonic stem (hES) cells in a manner that maximizes transfection by the included EditPro™ Stem Transfection Reagent to transfect genome editing constructs. The EditPro™ Stem is optimized for delivery of CRISPR/Cas9 editing via DNA Cas9 vectors, Cas9 mRNA with DS DNA (G-block), Cas9 mRNA with tracr/guideRNA, or Cas9 protein with tracr/guideRNA. It’s effective for delivery of TALEN and ZFN editing tools as well. With such high efficiency delivery, it’s not necessary to include an antibiotic selection in the editing design.

 

Fig. 1 Human iPS cells grown in G9™ Maintenance Medium on Vitronectin - Photo credit: MTI-Globalstem

Fig. 1 Human iPS cells grown in G9™ Maintenance Medium on Vitronectin – Photo credit: MTI-Globalstem

 

iPS or hES cells, when grown on the G9™ VTN included in the system in G9™ Maintenance Medium, maintain their pluripotency with a colony morphology which is that of a mono layer and ideal for even, high efficiency transfection when using EditPro™ Stem. The G9™ Versene is used as a gentle, easy, non-enzymatic method of passaging the cells.

Fig. 2 Transfection with a standard CRISPR/Cas9 EF1α-GFP DNA construct. NCRM-1 iPS cells transfected using EditPro Stem with a ~10.5 kb plasmid expressing Cas9 and GFP. Photo credit: MTI-Globalstem

Fig. 2 Transfection with a standard CRISPR/Cas9 EF1α-GFP DNA construct. NCRM-1 iPS cells transfected using EditPro Stem with a ~10.5 kb plasmid expressing Cas9 and GFP. Photo credit: MTI-Globalstem

 

Fig. 3 Indel analysis following transfection with EditPro Stem Transfection Reagent. Cells were transfected with Cas9 mRNA (modified)/ gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified) or Cas9 protein/gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified). Genome-modification was analyzed using the T7Endo 1 assay. Photo credit: MTI-Globalstem

Fig. 3 Indel analysis following transfection with EditPro Stem Transfection Reagent. Cells were transfected with Cas9 mRNA (modified)/ gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified) or Cas9 protein/gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified). Genome-modification was analyzed using the T7Endo 1 assay. Photo credit: MTI-Globalstem

 

EditPro™ Stem, when used to transfect iPS or hES cells as part of the G9™ Gene Editing System, results in the highest efficiency of transfection regardless of what type of substrate is being transfected. As an example, you can see transfection and expression efficiency  with a Cas9 DNA plasmid, and indel formation with transfection of mRNA with tracr/guide RNA or Cas9 protein with tracr/guide RNA. All methods give excellent editing results.

To resume, the benefits are…

  • High Efficiency Transfection of human pluripotent stem cells delivering DNA, RNA and/or protein
  • No need for antibiotic selection
  • Low toxicity and does not affect pluripotency
  • Easy to use – no special equipment required
  • Cost Effective – effective at low amounts of reagent resulting in low cost/well
  • Xeno-free, feeder-free culture of human pluripotent cells
  • Easy, gentle non-enzymatic passaging compatible with high throughput culture

Some extra reading? Take a look at the protocol..

There is a step-by-step protocol designed by MTI-Globalstem scientists, click here to take a look at it.

What’s in the box…

pluriq-g9-editing-500x500The components of the PluriQ™ G9™ Gene Editing System (also available separately) are:

  • EditPro™ Stem Transfection Reagent, 0.1 ml, optimized for the delivery of DNA, RNA and/or protein into pluripotent stem cells and tested specifically for delivery of CRISPR/Cas9 editing via DNA plasmid, mRNA Cas9 with tracr/guide RNA, or Cas9 protein with tracr/guide RNA. It is effective for TALEN and ZFN editing as well. Available separately as GST-2174 in the 0.25 ml size.
  • PluriQ™ G9™ Maintenance Medium Kit (GSK-9001), a defined, xeno-free, feeder-free medium used for expansion and maintenance of human ES and human iPS cells in vitro. Includes PluriQ™ G9™ Basal Medium (500 ml), PluriQ™ G9™ Supplement (50X), Human FGF-2, Human TGF-β1.
  • G9™ VTN Human Recombinant, 0.5 µg/ml (GSR-2070, 1 ml) for plate coating. This provides an ideal substrate which leads to the stem cells being more of a monolayer and a bit more spread out which enhances efficiency and evenness of transfection.
  •  G9™ Versene Solution (1x) (GSM- 1101, 100 ml) for passaging which allows for easy, non-enzymatic, gentle passaging of the cells and is compatible with scale-up to higher throughput cell culture.

Interested in learning more? Leave your questions and comments below, we’ll be pleased to answer!

Subscribe to e-Newsletters on your favourite topics
Interested in learning more about tools like this?

Subscribe to thematic newsletters on your favourite research topics. 
Written by Jean-François Têtu, PhD
Jean-François Têtu is Sales Manager at tebu-bio for the south of Europe and also for the UK and Eire. He also fervently defends and promotes anything from his home territory, the north of France.