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Applications:
• mRNA translation and degradation studies
• Protein biochemistry studies – synthesis, folding, processing, stability, localization, degradation
• mRNAtransferintoprimarycellswithoutgeneratinggeneticallymodifiedorganisms(GMO)
• GenomeengineeringbyRNA-onlyCRISPR/Castechnology
Highlyefficient,especiallyinprimarycells(e.g.,neuronsorHUVECs)andstemcells
ImmediatemRNAtranslation–protein synthesis detectable after15-30minutes
ImmediatemRNAdelivery–no endocytosis or lysosomal degradation
Extremely low cytotoxicity
in cooperation with
Fuse-It-mRNATheShortcutforProtein Expression
Primary cortical neurons + eGFP-mRNA
Primary myocytes + LifeAct®-TagGFP2-mRNA
ImmediatemRNADelivery
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.
MembraneFusion–ACompletelyNewTechnology
Cytotoxicity of Fuse-It-mRNA versus classical lipoplex-based method: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.
HighEfficiency,EveninPrimaryCells
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.
mRNA Transfer versus 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%.
ExtremelylowCytotoxicity
In contrast to classical 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.
Easy Handling
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.
Membrane Fusion DNA Transfection
6 h 6 h
2 h 2 h
Fuse-It-mRNAClassical lipoplex-based method
Fuse-It-mRNATheShortcutforProteinExpression
Fuse-It-mRNA Membrane Fusion
Cytoplasm
mRNA
mRNA
MEMBRANEFUSION
Fuse-It mRNA
LiposomalCarrier
TRANSCRIPTION
PlasmidTransfectionreagent
DNA lipid complex
DNA TRANSFECTION
RELEASE OF DNA
Endosome
ENDO-CYTOSIS
ENDO-CYTOSIS
mRNA lipid complex
mRNA TRANSFECTION
mRNA
RELEASE OF mRNA
Endosome
Transfectionreagent
TRANSLATION
Protein
MembraneFusion–TheDirectWaytoProteinExpression
ProteinSynthesisaftertheTransferofeGFP-mRNAinCHO-K1CellsUsingFuse-It-mRNA:
Tel.: +4989/5204617-0Fax: +4989/5204617-59 E-Mail: [email protected] © ibidi GmbH, V 1.0, 2016 / 04
ibidiGmbHAmKlopferspitz1982152Planegg/MartinsriedGermany
EfficiencyofmRNAExpressioninVariousCellTypesafterTreatmentwithFuse-It-mRNA:
OrderingInformation:
Cat.No. Description
60500 Fuse-It-mRNA, infrared fluorescent: 2 x 150 µl solution
60501 Fuse-It-mRNA, infrared fluorescent: 2 x 300 µl solution
ForadditionalFuse-Itproducts,please refer to:
www.ibidi.com
Forfurthercelltypes,pleasereferto:www.ibidi.com
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.
CellType Organism IncubationTimeEfficiencyofmRNA
Expression
PrimaryCells
Cortical Neurons (embryonal) rat 5-15 min 70-90%Hepatocytes human 15-25 min 60-80%Keratinocytes (foreskin) human 10-15 min 70-80%Monocytes / Macrophages human 10-15 min 30-50%Myocytes (embryonal) rat 15-25 min 40-60%Myofibroblasts (embryonal) rat 15-20 min 30-50%
StemCells
Cor.4U Myocytes (iPSC derived cardiomyocytes)
human 10‐20 min 80‐100%
Dopa.4U (iPSC derived dopaminergic neurons)
human 5‐10 min 70‐90%
iPSC human 10‐20 min 70‐90%Peri.4U (iPSC derived peripheral neurons)
human 5‐10 min 70‐90%
CellLines
CHO-K1 hamster 15-20 min 80-100%HeLa human 15-20 min 70-90%MCF10A human 10-15 min 80-100%RAW 264.7 rat 15-20 min 50-70%
2 h 20h 40h 90h
Formoreinformationonourproducts,pleasevisitusat:
www.ibidi.com
Fuse-It-mRNATheShortcutforProteinExpression