mRNA LifeAct-TagGFP2

Applications

• Transient transfer of LifeAct for fluorescence microscopy
• Non-toxic F-actin staining of living samples with superb biocompatibility
• Cytoskeleton organization:
  - Non-toxic actin visualization
  - Fluorescence staining
• Cytoskeletal dynamics:
  - Actin dynamics in cellular processes
  - Live cell imaging and microscopy

Specifications

Form	1 μg/μl in Tris buffer
Storage	–80 °C
TagGFP2 (Exmax / Emmax)	483 / 506 nm

Technical Features

• Highly pure mRNA—transcribed in vitro and optimized for high levels of LifeAct expression
• Mimics a fully processed, mature mRNA
• Vector backbone with 5'-ARCA cap (Anti-Reverse Cap Analog)
• High capping efficiency for high mRNA stability in the cytoplasm
• Strong Kozak sequence for efficient translation
• Poly(A) tail for high stability and robust protein expression
• GFP2 reporter for versatile fluorescence microscopy techniques
• Excellent signal-to-noise ratio
• Compatible with all classic mRNA transfection reagents, including Fuse-It-mRNA; see Application Note 56 (PDF) for details

Fuse-It-mRNA vesicles were filled with mRNA LifeAct-TagGFP2 and fused with human iPSC-derived cardiomyocytes. 16 hours after 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 for unmodified myocytes which show 50 to 80 contractions per minute.

mRNA LifeAct^®-TagGFP2 is a mammalian mRNA that encodes the LifeAct-TagGFP2 fusion protein. LifeAct, a 17-amino acid peptide, is derived from a protein found in Saccharomyces cerevisiae. It stains filamentous actin (F-actin) structures in living or fixed eukaryotic cells and tissues with excellent signal-to-noise ratio [Riedl et al., 2008]. mRNA LifeAct-TagGFP2 can be used for the fluorescent labeling of the actin cytoskeleton in various living cell types. For more information on the TagGFP2 reporter, please visit evrogen.com. The vector backbone also contains an ARCA cap, a strong Kozak sequence, and a poly(A) tail for efficient protein expression.

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Original LifeAct publication:
Riedl J, et al. Lifeact – a versatile marker for the visualization of F-actin. Nature Methods 5, 605-607 (2008)

LifeAct is a registered trademark of ibidi GmbH.
For license and product use information please refer to www.ibidi.com, Legal Notes.

Application Examples

Cardiomyocytes

F-actin visualization in primary myocytes expressing mRNA LifeAct-TagGFP2, seeded on a µ-Dish ESS 1.5 kPa.

Cervical Cancer Cells (HeLa)

HeLa cells transfected with mRNA LifeAct-TagGFP2 using Fuse-It-mRNA easy; 5 min incubation time.

Hepatocytes

Human hepatocytes transfected with mRNA LifeAct-TagGFP2 using Fuse-It-mRNA; 25 min incubation time.

Keratinocytes

Normal Human Epidermal Keratinocytes (nHEK) transfected with mRNA LifeAct-TagGFP2 using Fuse-It-mRNA; 25 min incubation time.

Method Comparison: Using LifeAct for F-Actin Visualization in Living Cells

	LifeAct-Plasmid With Torpedo DNA	LifeAct-mRNA With Fuse-It-mRNA	LifeAct-Protein With Fuse-It-P	LifeAct- Adenovirus	LifeAct- Lentivirus
Method Suitable for 1
Cell lines	+++	+++	+++	+++	+++
Primary cells	+	+++	++	+++	+++
Non-mammalian cells	-	+	+	-	-
Transfection Stability
Transient	Yes	Yes	Yes	Yes	No
Stable	Yes	No	No	No	Yes
Protocol and Analysis
Protocol simplicity	+++	++	++	+++	+++
LifeAct readout start	1–2 days	few hours	few minutes	1–2 days	1–2 days
LIfeAct readout duration	several days (infinity if stable)	several days	up to 1 day	several days	several days (infinity if stable)
Signal intensity	+++	+++	+	+++	+++
Extra Benefits
Prevention of overexpression artefacts	-	+	+++	+	+
Prevention of viral response	+++	+++	+++	-	-
Biocompatibility of transfer method	+	+++	+++	++	++
Actin functionality	+++	+++	+++	+++	+++
Safety Consideration
Biosafety Level	BSL1	-	-	BSL2	BSL2

¹ Not all of these combinations have been tested in all indicated cell types. We encourage you to use this table as a guideline to understand how the LifeAct system can be optimally applied.