APPLICATION NOTE Lipofectamine MessengerMAX Transfection Reagent

Optimizing cardiomyocyte transfection with Lipofectamine MessengerMAX reagent

IntroductionCardiovascular disease is the leading cause of mortality in the United States, and understanding the pathophysiology of the disease is a critical research area. In order to study the changes in signaling pathways that give rise to this class of diseases, in vitro experimental models are frequently employed. Transfection of cardiac muscle cells, or cardiomyocytes, has posed a particularly challenging problem, and achieving uniform protein expression at a high efficiency has been one of the biggest hurdles for reagent-based systems. Methodologies currently being employed include electroporation and viral overexpression, which come with a range of limitations including cost and time. Reagent-based transfection of DNA in nondividing primary cells is very inefficient due to ineffective nuclear entry and thus is not a viable tool—emphasizing the need for a reagent that can address this limitation. To simplify the process of overexpressing protein in cardiomyocytes, we developed the Invitrogen™ Lipofectamine™ MessengerMAX™ Transfection Reagent. Here we provide a simple protocol and examples of how Lipofectamine MessengerMAX reagent can be used for high-efficiency transfection of cardiomyocytes. The efficacy of this reagent system provides researchers with a superior overexpression system compared to the commonly used methods that include adenovirus and electroporation (Figure 1).

Figure 1. Recommended transfection methods for cardiomyocytes. As can be seen in the bright-field and GFP images above, the high transfection efficiency achieved using mRNA transfection makes Lipofectamine MessengerMAX reagent the recommended choice for use in cardiomyocytes. For researchers interested in other delivery methods, we’ve listed some additional potential solutions by cell type. The trade-offs of the alternative solutions are provided in Table 1.

Cell type

Alternative

transfection

methods

Primary cardiomyocytes

Recommended

solution

Lipofectamine MessengerMAX reagent

Cardiomyocyte stem cells

1. Adenovirus2. Electroporation3. Lipofectamine 3000

reagent

1. Adenovirus2. Electroporation

Posttransfection, bright-field

Posttransfection, GFP: 57% transfection efficiency

Protocol overview The Lipofectamine MessengerMAX reagent has been optimized for the delivery of mRNA, making it an ideal choice for delivery in isolated primary cardiomyocytes. Utilizing mRNA as a payload bypasses the need for nuclear entry, which greatly improves transfection effi ciency and subsequent protein expression, specifi cally in nondividing primary cells (Figures 2 and 3). A detailed protocol is outlined in Figure 4.

mRNA transfection

DNA transfection

Step 2.Endocytosis

into cell

Step 1.Attachment

to cell

Step 3.Escape

from endosome

Step 4.Nuclear

entry

Proteinexpression

Reagent–mRNA complex

Endosome

DNA

Protein

mRNA

Reagent–DNA complex

Nucleus

Figure 2. Faster protein expression with no risk of genomic integration. Transfection of mRNA with Lipofectamine MessengerMAX reagent typically results in faster protein expression with greater homogeneity of expression among the transfected cells. Additionally, delivery of mRNA does not require nuclear entry (step 4, DNA transfection), which eliminates the risk of genomic integration and makes transfection effi ency cell cycle–independent.

Expressionvector DNA

21 3

T7 ORF

Cloning option

PCR option

ARCA-capped mRNAwith poly(A) tail

Prepare DNA templatePrepare your DNA template with a T7 promoter. You may choose to clone your gene with a Gateway pcDNA-DEST40 vector or amplify it with PCR with T7-containing primers.

Generate mRNATranscribe your template DNA with the mMESSAGE mMACHINE T7 ULTRA Transcription Kit to generate mRNA for transfection.

Transfect mRNATransfect your mRNA with our simple Lipofectamine MessengerMAX transfection protocol.

Timeline Steps

Day

0

1

Seed cells to be 70–90% confluent at transfection

Day

1

2

Diluted MessengerMAX Reagent

Vortex 2–3 sec

3

Diluted mRNA

Prepare diluted mRNA master mix by adding mRNA to

Opti-MEM medium—mix well

4

5

Incubate

6

Add mRNA–lipid complex to cells

Day

2–4

7

Visualize/analyzetransfected cells

Gateway pcDNA-DEST40 Vector

SV40 ori

f1 ori

BGH pAP CMV

Am

pR

pUC oriSV40 pA

N

eoR

T7 attR1 attR2 6xHisccdBCmR V5 epitope

Dilute Lipofectamine MessengerMAX reagent in

Opti-MEM medium (2 tubes)—mix well

Add diluted mRNA to each tube of diluted

Lipofectamine MessengerMAXreagent (1:1 ratio)

Figure 3. Workfl ow for mRNA transfection using Lipofectamine MessengerMAX reagent.

High-effi ciency transfection of cardiomyocytesThe Lipofectamine MessengerMAX reagent demonstrates high performance in freshly isolated, primary neonatal rat ventricular cardiomyocytes, demonstrating high effi ciency and homogeneity of transfection, and signifi cantly outperforming leading DNA transfection reagents. In addition to high transfection effi ciency, the reagent has low toxicity to the primary cells, as can be seen by the integrity of the culture system 24 hours posttransfection (Figure 5). Optimization of transfection effi ciency, depending on the experimental need of the researcher, can be achieved by modulating plating density, with minimal effect on cell viability (Figure 6).

Figure 5. Superior transfection achieved in cardiomyocytes using mRNA transfection vs. DNA transfection. (A) Phase-contrast and fl uorescence images are shown for comparison. Fluorescence imaging shows GFP-positive primary cardiomyocytes 24 hours following transfection using either Invitrogen™ Lipofectamine™ 3000 reagent + GFP plasmid DNA, or Lipofectamine MessengerMAX reagent + GFP mRNA. (B) Summary of fl ow cytometry analysis of transfection effi ciency demonstrates the high effi ciency of transfection achieved using mRNA delivery.

Figure 4. Step-by-step protocol for transfecting cardiomyocytes using Lipofectamine MessengerMAX reagent.

Figure 6. Optimization of mRNA expression. Fluorescence images of primary cardiomyocytes 24 hours after transfection with mRNA + Lipofectamine MessengerMAX reagent, demonstrating increasing transfection effi ciency with increasing plating density of the cardiomyocytes. This system overcomes a variety of experimental design constraints, as researchers can still achieve high-effi ciency transfection with minimal toxicity at lower plating densities.

Phase contrast GFP

Lip

ofe

ctam

ine

30

00

reag

ent

+ G

FP

pla

smid

DN

A

Lip

ofe

ctam

ine

Mes

sen

ger

MA

X

reag

ent

+ G

FP

mR

NA

Timeline Steps

Day

0

1

Seed cells to be 70–90% confl uent at transfection

Day

1

2

Diluted Lipofectamine

MessengerMAX reagent

Dilute Lipofectamine MessengerMAX reagent in

Opti-MEM medium (2 tubes)—mix well

3 10Incubate

4

Diluted mRNA

Prepare diluted mRNA master mix by adding mRNA to

Opti-MEM medium—mix well

5

Add diluted mRNA to each tube of diluted Lipofectamine

MessengerMAX reagent (1:1 ratio)

6

Incubate

7

Add mRNA–lipid complex to cells

Day

2–3

8

Visualize and analyze transfected cells

Procedure details (two-reaction optimization)Component 24-well

Adherent cells 0.5–2 x 105

Opti-MEM medium 25 μL x 2

Lipofectamine MessengerMAX reagent 0.75 and 1.5 μL

Incubate diluted Lipofectamine MessengerMAX reagent in Opti-MEM medium for 10 min at room temperature

Opti-MEM medium 50 μL

mRNA (0.5–5 μg/μL) 1 μg

Diluted mRNA 25 μL

Diluted Lipofectamine MessengerMAX reagent 25 μL

Incubate for 5 min at room temperature

Component (per well) 24-well

mRNA–lipid complex 50 μL

mRNA 500 ng

Lipofectamine MessengerMAX reagent 0.75 and 1.5 μL

Incubate cells for 1–2 days at 37°C, then analyze transfected cells.

Vortex 2–3 sec

A

B

0

10

20

30

40

50

60

Lipofectamine MessengerMAX reagent + mRNA

Lipofectamine 3000 reagent

+ GFP plasmid

Negative control

Tra

nsfe

ctio

n ef

ficie

ncy

(%)

70

Cells seeded 7.5 x 104 8.5 x 104 1.1 x 105 1.75 x 105

35% 45% 51% 57%Effi ciency

See all of our application notes at thermofisher.com/transfectionbasics

Find out more about Lipofectamine MessengerMAX reagent at thermofisher.com/messengermax

For Research Use Only. Not for use in diagnostic procedures. © 2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. COL31436 0117

Table 1. Comparison of different transfection techniques.

Transfection technique

Expression efficiency

Cost effectiveness

Cell viability

Experimental ease of use

Electroporation ++ + + ++

Adenoviral overexpression +++ ++ +++ +

Leading DNA transfection reagent

+ ++ ++ +++

Lipofectamine MessengerMAX reagent

+++ +++ +++ +++

ConclusionsAs demonstrated here, Lipofectamine MessengerMAX reagent offers a highly effective solution for protein overexpression in difficult-to-transfect primary cell models, providing several benefits including ease of use and high efficiency. This reagent is very gentle on isolated primary cardiomyocytes and differentiated cardiomyocytes, compared to other commonly used transfection methods. While several methodologies exist for transfecting cardiomyocytes, we recommend Lipofectamine MessengerMAX reagent as the primary gene delivery solution.

Additional applications for cardiomyocyte modelsTransfection of human induced pluripotent stem cell (iPSC)-derived cardiomyocytes is an important experimental platform for studying cardiovascular disease. Differentiated iPSCs comprise a very sensitive cell culture system and offer a physiologically relevant model that can be used for translational research. Using mRNA with Lipofectamine MessengerMAX reagent, transfection efficiency greater than >30% was achieved in these cells as seen from the fluorescence images taken 18 hours following transfection with GFP mRNA (Figure 7).

Figure 7. High-efficiency mRNA transfection in iPSC-derived cardiomyocytes using Lipofectamine MessengerMAX reagent.

Trade-offsA comparison of the main transfection techniques that are commonly used for cardiomyocytes highlights the benefits and drawbacks of each system. Electroporation requires physical disruption of the cell, which results in high cell mortality. It also requires specialized instrumentation and corresponding consumables, which can be expensive. Adenoviral overexpression is a highly efficient technique and frequently used for cardiomyocytes; however, there are several drawbacks associated with the use of this methodology. In addition to requiring adherence to specific laboratory guidelines and considerations for handling and use, generating adenoviral constructs is a time-consuming and expensive process. While high expression efficiency is achievable using adenovirus, the ease of use of this method in experimental design is limited to making a separate virus for each individual protein, which can be very time-consuming and high in cost. The practicality of making a library of constructs for mRNA or DNA transfection is more feasible than generating individual virus constructs for each experimental target. Table 1 compares mRNA transfection using Lipofectamine MessengerMAX reagent to the more commonly used transfection techniques that are used for cardiomyocytes.