Tranfsection of Simplicon™ RNAs has been validated using the RiboJuice™ mRNA Transfection Kit and Lipofectamine® MessengerMAX™ Transfection Reagent. Amounts of RNAs and transfection reagents may vary depending on the target cells. Set up different RNA: transfection reagent ratios.
a. Option 1: Add 200 ng/mL of B18R protein (MilliporeSigma GF156 or Sigma GF197) in medium. Pre-treatment of B18R protein may support the neutralization of IFN responses. Incubate cells at 37 °C in a CO2 incubator for 10-20 minutes with B18R protein.
b. Option 2: No serum condition increases the transfection efficiency. However, it is possible to use 1-10% serum depending upon cell types.
If using MessengerMAX™ Transfection Reagent (ThermoFisher LMRNA001): |
---|
If using RiboJuice™ mRNA Transfection Kit (Part No. TR-1013): |
---|
B) Reverse Transfection Protocol
For some cells (i.e. HepG2) reverse transfection may be more efficient.
C) Electroporation for primary T cells (human)
Electroporation is an alternative way to introduce Simplicon™ RNA into difficult to transfect cells such as primary human T cells (activated and expanded from peripheral blood mononuclear cells (PBMCs)). Using this protocol, it is possible to achieve 20-70% electroporation efficiency of Simplicon™ TagGFP2 RNA to primary human T cells.
In the Simplicon™ RNA Expression System, a new IFN suppressor, E3L, was introduced as a polycistronic B18R-E3L RNA to suppress the IFN responses at RNA transfection and also incorporated into Simplicon™ RNA itself to suppress the IFN responses during RNA replication. B18R-E3L RNA worked better than B18R RNA at the transfection with Simplicon™ TagGFP2 (Figure 1A). Addition of E3L into Simplicon™ RNA increased the expression of Simplicon™ TagRFP in sustained expression (Figure 1B). Simplicon™ RNA can be introduced with RNA transfection or RNA electroporation in many types of cells as indicated (Figure 1C).
B18R-E3L RNA is also available for mRNA transfection to suppress the IFN responses. B18R-E3L RNA worked better than B18R-RNA in mRNA transfection (Figure 2A). Suppression of IFN responses at mRNA transfection enables the repeated transfection of mRNA (Figure 2B).
Figure 1.(A) E3L increased SimpliconTM RNA expression levels. BJ human foreskin fibroblasts were co-transfected with SimpliconTM TagGFP2 and B18R RNA or B18R-E3L RNA. (B) E3L worked for continuous expression of SimpliconTM RNA. SimpliconTM TagRFP or TagRFP Simplicon (E3L) was co-transfected with B18R-E3L RNA and cultured with medium containing B18R protein and puromycin for 14 days. RFP expressing cells were imaged on Day 12, and analyzed by FACS on Day 14. (C) SimpliconTM RNA can be transfected into a wide variety of cell types. Simplicon™ TagGFP2 RNA and B18R-RNA were co-transfected with Human iPSCs, LX2 human hepatic stellate cell line (MilliporeSigma SCC064), human mesenchymal stem cells (MSCs, MilliporeSigma SCC034) by MessengerMAX™ transfection reagent. For human primary T cells (PBMCs stimulated with CD3/CD28), electroporation method was used.
Figure 2. B18R-E3L or B18R RNA for mRNA transfection. (A) BJ human foreskin fibroblasts were co-transfected with TagGFP2 mRNA and B18R RNA or B18R-E3L RNA. (B) B18R or B18R-E3L RNA co-transfection enables repeated transfection of mRNA. BJ cells were transfected with TagGFP2 mRNA Plus/Minus B18R RNA or B18R-E3L RNA at 1st transfection (upper panel, 1st transfection). Next day, TagRFP mRNA was transfected into the same cells, and imaged for RFP expression one day after the transfection (bottom panel, 2nd transfection). The mRNAs for TagGFP2 and TagRFP were synthesized without modified nucleotides.
To continue reading please sign in or create an account.
Don't Have An Account?