- mRNA translation and degradation studies
- Protein biochemistry studies: synthesis, folding, processing, stability, localization, degradation
- mRNA transfer into primary cells without generating genetically modified organisms (GMO)
- Genome engineering by RNA-only CRISPR/Cas technology
- Lipofection-independent transfer of mRNA into living cells
- mRNA transfer completed within 5–20 minutes
- No endocytosis involved
- No lysosomal degradation needed
- No gene transfer to the nucleus
- Protocol optimized for the transfer of functionally capped and polyadenylated mRNA
- No Biosafety Laboratory Level 1 or 2 necessary
- Excellent biocompatibility with low cytotoxicity
|ExMax/EmMax||750/780 nm (infrared)|
|Fusogenic Solution (FS)||2 vials|
|Neutralization Buffer (NB)||2 vials|
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 delivery methods, cells do not internalize the mRNA by endocytosis.
Lipoplex-Based DNA Transfection
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.
Lipoplex-Based mRNA Transfection
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
|Bone marrow-derived dendritic cells (BMDCs)||murine||20 min||70–80%|
|Cortical neurons, embryonic||rat||5–15 min||70–90%|
|Fibroblasts, foreskin||human||10–15 min||60–80%|
|Myocytes, embryonic||rat||15–25 min||40–60%|
|Myofibroblasts, embryonic||rat||15–20 min||30–50%|
|nHEK, foreskin||human||5–15 min||70–80%|
|ZNS cells, unpurified||rat||5–15 min||80–100%|
|HoxB8-derived dendritic cells||murine||5 min||90–100%|
|N/TERT-1 (keratinocytes, immortalized by expression of TERT)||human||10 min||80–90%|
|RAW 264.7||rat||15–20 min||50–70%|