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EditPro? Stem Transfection Reagent

EditPro? Stem Transfection Reagent Product Code: GST-2174 Availability: In Stock

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EditPro? Stem Transfection Reagent is optimized for genome editing in human pluripotent stem cells and neural stem cells. It is possible to co-transfect different substrates providing the flexibility needed for genome editing experiments.  For example EditPro? Stem provides efficient transfection of large DNA plasmids such as CRISPR/Cas9 vectors, RNA such as Cas9 mRNA co-transfected with tracr/guide RNA, or protein and RNA such as Cas9 protein with tracr/guide RNA. It also results in high efficiency transfection with TALENs and ZFN gene editing tools.
EditPro? Stem Transfection Reagent is easy to use and can work with various human pluripotent stem cell culture conditions (media and matrices). The efficiency is maximized when transfecting with the cells in adherence, however, when used in conjunction with the PluriQ? G9? Maintenance Medium Kit with the cells plated on G9? VTN Human Recombinant as matrix as provided in the PluriQ? G9? Gene Editing System. The reason for the boost using this culturing format is that the cells grow more as a monolayer and therefore are more accessible to transfection resulting in even and high efficiency transfection with low toxicity. 
iPS cells grown in G9 medium on vitronectin
Fig. 1 iPS Cells grown in PluriQ? G9? Maintenance Medium on G9? VTN Human Recombinant as matrix.
EditPro? Stem when used to transfect iPS or hES cells as part of thePluriQ? 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.
ipsc dna transfection

Fig. 2 High Efficiency Transfection with a standard CRISPR/Cas9 EF1a-GFP DNA construct. NCRM-1iPS cells transfected using EditPro Stem with a ~10.5 kb plasmid expressing Cas9 and GFP.

ipsc mrna and rnp transfection

Fig. 3 Transfection with EditPro? Stem Transfection Reagent Results in Effective Editing.  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.

To allow flexibility in choice of pluripotent stem cell culture system, such as plating on other matrices or in other commercial media, we optimized transfection in suspension using a "reverse transfection" technique, for maximum transfection of all the cells in culture. Adherent cells grown in colonies on these matrices do not evenly transfect due to limited access of the reagent to the inner cells of the colony. Figure 3 illustrates how co-transfection of modified GFP mRNA and Cas9 RNP using the EditPro Stem reagent consistently produces >90% transfection efficiency as monitored by GFP in stem cells plated on vitronectin in PluriQ? G9? Maintenance Medium and on Geltrex in mTeSR?1.

suspension transfection ipsc

Fig. 4 Transfection in Suspension: Expression of eGFP mRNA co-delivered with Cas9 Protein, tracrRNA and crRNAs Emx1 in human ESCs plated on A. vitronectin in PluriQ G9 Maintenance Medium or B. Geltrex in mTeSR

Using the transfection in suspension protocol, the modified Cas9 mRNA, tracrRNA and a modified GFP mRNA were co-transfected. The cells were then analyzed by T7Endo1 assay to look at the effectiveness of editing.

mRNA suspension transfection iPSC

Fig. 5 Cas9 mRNA Transfection into iPSC Cells with EditPro? Stem:   200,000 cells were transfected in suspension using EditPro? Stem with 250 ng Cas9 mRNA (modified)/ gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified). Genome-modification was analyzed using the T7Endo 1 assay.

suspension transfection H9

Fig. 6 Cas9  Protein (RNP) Transfection of  human iPS and ES cells with EditPro? Stem:   200,000 cells were transfected in suspension using EditPro? Stem with Cas9 protein/gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified). Genome-modification was analyzed using the T7Endo 1 assay

EditPro? Stem provides superior delivery into neural stem cells. Using iPS-derived human neural stem cells and the protocol for transfection in suspension with subsequent plating on Geltrex in NeuralX? NSC Medium Supplemented with GS22? we see >90% efficiency by GFP reporter and excellent editing using either Cas9 mRNA or Cas9 Protein (RNP) as evaluated by T7Endo 1 assay.

GFP in transfected NSC

indel after transfection of NSC

Fig. 7 Gene Editing by co-Transfection using EditPro? Stem Transfection Reagent in Neural Stem Cells: Neural Stem cells were transfected in suspension using EditPro? Stem with 250 ng 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). GFP was observed after 24 hours. Genome-modification was analyzed using the T7Endo 1 assay.

Features:

  • High Efficiency – delivering DNA, RNA and/or protein

  • 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


Technical Data



Publications

  • Kehler, J., Greco, M., Martino, V., Pachiappan, M., Yokoe, H., Chen, A., Yang, M., Jessee, J., Gotte, M., Milanesi, L., Albertini, A., Bellipanni, G., Zucchi, I., Reinbold, R. A. and Giordano, A. (2016), RNA-Generated and Gene-Edited Induced Pluripotent Stem Cells for Disease Modeling and Therapy. J. Cell. Physiol. 2016 Sep 15. doi: 10.1002/jcp.25597. [Epub ahead of print]


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