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Fuse-It-mRNA

Fuse-It-mRNA View larger

A fusion reagent for rapid transfection of mRNA into the cytoplasm of living cells

  • Immediate mRNA translation—protein synthesis detectable after 15–30 minutes
  • Highly efficient, especially in primary cells (e.g., neurons or HUVECs) and stem cells
  • Fastest mRNA delivery—no endocytosis or lysosomal degradation; no gene transfer into the nucleus needed
  • Extremely low cytotoxicity
  • Ready to use

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infrared fluorescent

Product Details

Applications:

  • 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

Technical Features:

  • 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

Specifications:

Form Solution
Concentration 6 mM
Storage –20 °C
ExMax/EmMax 750/780 nm (infrared)

Kit Components:

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.

Fuse-It-mRNA

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.

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.

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.

Fuse-It-mRNA

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 in Various Cell Types after Treatment with Fuse-It-mRNA:

Cell Type

Organism

Incubation Time

Efficiency of mRNA Expression

Primary Cells

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%
Hepatocytes human 15–25 min 60–80%
HUVEC human 10–20 min 50–70%
Macrophages/monocytes human 10–15 min 30–50%
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%

Stem Cells

iPSC human 10–20 min 70–90%
MSC human 5–15 min 60–80%

Cell Lines

A549 human 20–25 min 80–100%
CHO-K1 hamster 15–20 min 80–100%
HeLa human 15–20 min 70–90%
HL-1
mouse 15–20 min 5–10%
HoxB8-derived dendritic cells murine 5 min 90–100%
HT-1080 human 15–20 min 80–100%
HUH-7 human 10–15 min 70–90%
Jurkat human 1–2 min 20–50%
L929 mouse 15–20 min 70–90%
MCF-10A human 10–15 min 80–100%
MCF-7 human 15–20 min 70–90%
MDA-MB-231 human 10–15 min 70–90%
MDCK dog 20–25 min 20–40%
NIH-3T3 mouse 15–20 min 70–90%
N/TERT-1 (keratinocytes, immortalized by expression of TERT) human 10 min 80–90%
Rat1 rat 5–10 min 80–100%
RAW 264.7 rat 15–20 min 50–70%
THP-1 human 1–2 min 40–70%

Primary Cells

Cortical neurons, embryonic
Organism: rat
Incubation Time: 15 min
mRNA eGFP

Fibroblasts, foreskin
Organism: human
Incubation Time: 15 min
mRNA eGFP

Hepatocytes
Organism: human
Incubation Time: 25 min
mRNA LifeAct-TagGFP2

Macrophages/monocytes
Organism: human
Incubation Time: 15 min
mRNA eGFP

HUVEC
Organism: human
Incubation Time: 20 min
mRNA eGFP

HUVEC
Organism: human
Incubation Time: 20 min
mRNA LifeAct-TagGFP2

Myocytes, embryonic
Organism: rat
Incubation Time: 25 min
mRNA eGFP

Myofibroblasts, embryonic
Organism: rat
Incubation Time: 20 min
mRNA eGFP

nHEK, foreskin
Organism: human
Incubation Time: 20 min
mRNA eGFP

nHEK, foreskin
Organism: human
Incubation Time: 20 min
mRNA LifeAct-TagGFP2

ZNS cells, unpurified
Organism: rat
Incubation Time: 15 min
mRNA eGFP

Stem Cells

iPSC
Organism: human
Incubation Time: 20 min
mRNA eGFP

MSC
Organism: human
Incubation Time: 5–15 min
mRNA eGFP

Cell Lines

CHO-K1
Organism: hamster
Incubation Time: 20 min
mRNA eGFP

CHO-K1
Organism: hamster
Incubation Time: 20 min
mRNA mCherry

HeLa
Organism: human
Incubation Time: 20 min
mRNA eGFP

HL-1
Organism: mouse
Incubation Time: 20 min
mRNA eGFP

HT-1080
Organism: human
Incubation Time: 20 min
mRNA eGFP

HUH-7
Organism: human
Incubation Time: 15 min
mRNA eGFP

Jurkat
Organism: human
Incubation Time: 12 min
mRNA eGFP

L929
Organism: mouse
Incubation Time: 15 min
mRNA eGFP

MCF-10A
Organism: human
Incubation Time: 15 min
mRNA eGFP

MCF-7
Organism: human
Incubation Time: 20 min
mRNA eGFP

MDA-MB-231
Organism: human
Incubation Time: 15 min
mRNA eGFP

NIH-3T3
Organism: mouse
Incubation Time: 20 min
mRNA eGFP

N/TERT-1
(keratinocytes, immortalized by expression of TERT)
Organism: human
Incubation Time: 10 min
mRNA eGFP

Rat1
Organism: rat
Incubation Time: 10 min
mRNA eGFP

RAW 264.7
Organism: rat
Incubation Time: 20 min
mRNA eGFP

THP-1
Organism: human
Incubation Time: 12 min
mRNA eGFP

Supporting Material

Movies

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.

User Comments

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

http://www.a-star.edu.sg/imb

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

http://www.wbex.med.uni-muenchen.de/

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

www.pharmbiol.cup.uni-muenchen.de/staff/group-leaders/zahler/index.html

Moritz Schneider, Ludwig-Maximilians-University, München, Germany

"We could successfully apply Fuse-It-mRNA for transfection of A549 and MDCK cells. The protocol was very easy to follow, and the transfection efficiency exceeded our expectations!”

Moritz Schneider
Ludwig-Maximilians-University
Munich
Germany

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