A Gene Editing System for Stem Cells
The PluriQ™ G9™ Gene Editing System is a complete system for culturing and transfecting human pluripotent stem cells for gene editing. The 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. 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/guide RNA, or Cas9 protein with tracr/guideRNA. It is effective for delivery of TALEN and ZFN editing tools as well. With such high efficiency delivery it is not necessary to include an antibiotic selection in the editing design.
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 monolayer 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. 1 Human iPS cells grown in G9™ Maintenance Medium on Vitronectin
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, we show 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.
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.
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.
The 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.
- 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
Expand from single cells for clonal selection while maintaining pluripotency using PluriQ™ G9™ Cloning Medium (100ml).
|Recommended Use||Culture and transfection for genome editing of human ESC or iPSC.|
|Storage||Store refrigerated at 2-8°C: ● PluriQ™ G9 Basal Medium ● EditPro™ Stem Transfection Reagent ● G9™ Versene Solution Store -20°C (non frost-free freezer): ● PluriQ™ G9 Supplement ● Human FGF-2 ● Human TGF-β1 Store -80°C ● G9™ VTN Human Recombinant|
|Quality Testing||See individual components. Tested functionally together.|
|Shipping||Room Temp: ● PluriQ™ G9 Basal Medium ● EditPro™ Stem Transfection Reagent ● G9™ Versene Solution Dry Ice: ● PluriQ™ G9 Supplement ● Human FGF-2 ● Human TGF-β1 ● G9™ VTN Human Recombinant|
|Other||For Research Use Only - Not intended for human or animal diagnostic or therapeutic uses|
PluriQ™ G9™ Gene Editing System
- Product Code: GSK-9003
- Availability: In Stock