Fuse-It-mRNA Results in Highly Efficient Protein Synthesis, Even in Primary Cells.
Fuse-It-mRNA vesicles were filled with eGFP-mRNA and fused with normal human epidermal keratinocytes (nHEK) for 10 minutes. Two and a half hours after eGFP-mRNA transfer, 50% of the cells express eGFP, and after eight hours almost 100% express eGFP.
Membrane Fusion - The Direct Way to Protein Expression:
Immediate mRNA Delivery
The Fuse-It liposomal carrier, which includes the mRNA, simply fuses with the cell membrane and then releases the mRNA directly into the cytoplasm. mRNA translation starts immediately, without the interfering processes of endocytosis, lysosomal degradation, or mitosis. Unlike classical lipoplex-based delivey methods, cells do not internalize the mRNA by endocytosis.
Fuse-It-mRNA results in extremely fast mRNA expression. Six hours after eGFP-mRNA transfer by membrane fusion of CHO-K1 cells with Fuse-It-mRNA, nearly 100% of the cells express eGFP, whereas the number of eGFP-expressing cells after plasmid-transfection is below 10%.
High Efficiency, Even in Primary Cells
Based on the charge of natural cell membranes, Fuse-It-mRNA liposomes are able to effectively fuse with most cell types. Cell lines, non-proliferating cells (e.g., neurons), and a broad spectrum of difficult-to-transfect primary cells can directly translate the mRNA in the cytosol. Membrane fusion with Fuse-It-mRNA results in fast and highly efficient protein expression with no risk of genomic integration.
The Fuse-It-mRNA protocol is easy to use because the ratio between fusion reagent and mRNA remains constant in each experiment. The reagent can be used without restrictions because no genetically modified organisms are generated.
Extremely low Cytotoxicity
In contrast to classic lipoplex-based methods, Fuse-It-mRNA requires only brief incubation times - between 5 and 20 minutes for maximal nucleotide transfer rates. Furthermore, low amounts of liposomal lipids result in maximal mRNA transfer, without the need for chemical compounds for endosomal release. This feature protects sensitive cells from potential toxic effects of carrier reagents.
In contrast to classical lipoplex-based methods, Fuse-It-mRNA fusion does not harm cells. Sixteen hours after eGFP-mRNA transfer by membrane fusion of primary human epithelial cells (nHEK) with Fuse-It-mRNA, the vitality of the cells is high, whereas significant cell death occurred in the cells treated with classical lipoplex-based methods.
Protein Synthesis after the Transfer of eGFP-mRNA in CHO-K1 Cells Using Fuse-It-mRNA:
Within 2 hours of starting the experiment, the eGFP is already detectable in the cells. The eGFP amount increases and remains stable for more than 3 days (90 hours).
NOTE: Using functionally capped and polyadenylated mRNAs will achieve the best results.
Efficiency of mRNA Expression in Various Cell Types after Treatment with Fuse-It-mRNA:
Fuse-It-mRNA results in extremely fast mRNA translation into proteins.
Fuse-It-mRNA vesicles were filled with eGFP-mRNA and fused with CHO-K1 cells. Two hours after eGFP-mRNA transfer, 50% of the cells express eGFP, and after six hours almost 100% express eGFP.
Fuse-It-mRNA does not influence the contraction rates of cardiomyocytes.
Fuse-It-mRNA vesicles were filled with LifeAct®-TagGFP2-mRNA and fused with human iPSC-derived cardiomyocytes (Cor.4U). 16 hours after LifeAct®-TagGFP2-mRNA transfer, the contractions per minute were measured. The myocytes showed contraction rates of about 70 beats per minute. This value is in the normal range of unmodified myocytes which show 50 to 80 contractions per minute.
Christine Neumann, Institute of Medical Biology, A*STAR, Singapore
"The Fuse-It-mRNA kit was easy to use and worked very well for the N/TERT-1 immortalized skin cells I transfected. At the time of transfection, keratinocytes were 70% confluent and showed excellent viability after transfection. The GFP became visible two hours after transfection and lasted for more than one week while keratinocytes were undergoing differentiation. With an efficiency of more than 50% positive cells and unaffected cell morphology, it is an unbeatable alternative to electroporation or lipofection techniques used for transient transfection with our difficult to transfect keratinocytes."
Christine Neumann Institute of Medical Biology A*STAR Singapore
Louise Ince, Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilian-University, Munich, Germany
"I used the Fuse-It-mRNA kit to label mature murine bone marrow-derived dendritic cells with GFP and was very happy with the results. The kit was quick and easy to use, and is an excellent non-viral transfection method for these cells with only a sonicator required.”
Louise Ince Walter Brendel Centre of Experimental Medicine Ludwig-Maximilians-University Munich Germany
Stefan Zahler, Pharmaceutical Biology, Ludwig-Maximilians-University, Munich, Germany
“Good cell viability and good transfection efficiency, at the same time, are hard to get with HUVECs. Shuttling mRNA directly into the cells with Fuse-It-mRNA gave a very good efficiency (> 60% positive cells), and left the cells in an excellent state. This approach might be the long-desired alternative to using plasmids in cells which are hard to transfect.”
Stefan Zahler Pharmaceutical Biology Ludwig-Maximilians-University Munich Germany