In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

Summary

In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in cells.

Abstract

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.

Introduction

In cells, the transcription of messenger RNA (mRNA) and the following translation of mRNA into desired proteins ensure the proper functioning of cells. Hereditary or acquired genetic disorders can lead to insufficient and dysfunctional synthesis of proteins and cause severe diseases. Thus, a new therapeutic approach to induce the production of missing or defective proteins is the exogenous delivery of synthetic modified mRNA into cells, which codes for the desired protein. Thereby, cells are triggered to synthesize functional proteins, which they normally cannot produce or would naturally not need. Using this approach, genetic disorders can be corrected by introduction of mRNA that codes for the defective or missing protein ¹. The mRNA therapy can also be used for vaccination to synthetize protein antigens, which are expressed by tumor cells or pathogens. Thereby, host immune system can be activated to effectively eliminate tumor cells or prevent infections ^2,3. Furthermore, in recent years, mRNA was successfully used to generate induced pluripotent stem cells (iPSCs). For this purpose, fibroblasts were transfected with mRNAs to induce the expression of reprogramming factors ^4-6 and to convert them in iPSCs with an enormous potential in regenerative medicine.

Previously, the use of conventional mRNA was associated with low stability and strong immunogenicity. Thus, clinical applications of conventional mRNAs were limited. However, the replacement of cytidine and uridine by 5-methylcytidine and pseudouridine within the mRNA molecule by Kariko and colleagues rendered mRNA molecules stable in biological fluids and dramatically reduced immune activation ^7-10, which now allows the clinical applicability of modified mRNAs.

Using synthetically produced modified mRNAs, desired gene transcripts can be temporarily delivered in vivo ¹¹ or in vitro to induce protein expression. The introduced mRNA is translated under physiological conditions by the cellular translation machinery. Due to lack of integration into the host cell genome compared to viral gene therapy vectors, the risk of oncogenesis is prevented ^12,13. Thus, therapy using modified synthetic mRNA will get better clinical acceptance in the future.

Here, we describe a detailed protocol for production of modified mRNA (Figure 1), transfection of cells with mRNA and the evaluation of protein expression in transfected cells.

Protocol

1. Augmentation of Plasmids Containing Required Coding DNA Sequences (CDS)

1. Pre-warm SOC medium, which is included in the transformation kit, to room temperature and the LB agar plates containing 100 µg/ml ampicillin to 37 °C. Equilibrate the water bath to 42 °C.
2. Thaw one vial of chemically component E. coli on ice.
3. Add 1-5 µl of the plasmid (10 pg to 100 ng) containing the CDS into the vial of chemically component E. coli and mix gently. Do not mix the cells by pipetting. After adding plasmids, mix by tapping the tube gently.
4. Incubate the mixture on ice for 30 min.
5. Heat-shock the E. coli for 30 sec at 42 °C in a water bath without shaking.
6. Place the vial on ice for 2 min.
7. Add 250 µl of pre-warmed SOC medium to the E. coli and shake the bacteria horizontally at 300 rpm for 1 hr at 37 °C in a bacterial incubator.
8. Spread 100 µl and 150 µl from transformation mix on pre-warmed LB agar plates, invert the plates and incubate overnight at 37 °C.
9. After colonies become visible, inoculate a single colony from each plate into a 15 ml culture tube containing 5 ml LB medium with 100 µg/ml ampicillin. Incubate them overnight at 37 °C with shaking at 300 rpm until culture is in late log or stationary phase.
10. For long-term storage of transformed E. coli, pipet 75 µl of 100% glycerol into the cryovial. Add 225 µl of the bacterial culture (frozen stock will contain 25% glycerol), mix well and store the cryovial at -80 °C.
11. Isolate the plasmids containing the required CDS using a plasmid purification kit according to manufacturer's instructions.
12. Determine the plasmid concentration using a spectrophotometer.
13. Prepare aliquots of 20 µl and store them at -20 °C for prolonged time.

2. Amplification of Plasmid Inserts and Adding of Poly T-tail by Polymerase Chain Reaction (PCR)

1. NOTE: To obtain the DNA template for the in vitro transcription (IVT), the CDS of interest, here eGFP, is amplified using PCR. Simultaneously, a poly T-tail of 120 thymidines (T) is added to the insert by using a reverse primer with a T₁₂₀ extension. Thereby, the generated mRNAs obtain a poly A-tail with a defined length after the IVT.
2. Prepare PCR mixture as detailed in Table 1.
3. Mix the reaction mix thoroughly. NOTE: PCR product can be used in 4-5 IVT reactions. To increase the DNA template amount for IVT, total volume of the PCR mixture can be increased. Several PCR tubes each containing 100 µl PCR mixture can be used to obtain a sufficient yield of PCR product.
4. Place the PCR tubes in a thermocycler and run the PCR using the PCR cycling protocol (Table 2).
5. Clean up the PCR reaction using a PCR purification kit according to manufacturer's instructions and elute the DNA using 20 µl nuclease-free water.
6. Measure the concentration of the DNA using a spectrophotometer. NOTE: The detected DNA concentration is approximately 300 µg/ml. Thus, the expected total amount should be ~6 µg. The amount of DNA for other proteins can differ depending on the length of CDS, amount of plasmid used for PCR, annealing temperature of primers, and cycle number.
7. Freeze the DNA at -20 °C for a long time or use it directly for IVT.

3. Control Step: Quality of PCR Product

1. Check the quality of the PCR product by DNA gel electrophoresis.
2. Mix the desired amount of agarose with 1x TBE in a flask. For a 1% agarose gel, add 0.5 g of agarose to 50 ml of 1x TBE.
3. Microwave until the agarose is dissolved. Do not let it boil. Ensure that the agarose is completely in solution.
4. Add 5 µl nucleic acid gel stain to 50 ml agarose gel.
5. Pour the gel into an agarose gel box, set the comb, and wait approximately 1 hr until the gel solidified.
6. Remove the comb and place the gel box in a gel tray.
7. Mix 2 µl (200 ng) of 80-10,000 bp DNA-Ladder and 200 ng of PCR product each with 2 µl 6x loading buffer in a total volume of 12 µl.
8. Carefully load DNA samples into the wells of the agarose gel.
9. Using 1x TBE as running buffer, run the agarose gel at 100 V for 1 hr.
10. Visualize the DNA bands on an imaging system (Figure 2).

4. In Vitro Transcription (IVT)

1. NOTE: After PCR, the plasmid inserts are amplified and a poly T-tail is added. During the IVT, genetic information is transcribed from DNA to RNA. The generated mRNA transcript is used to produce proteins in cells.
2. Clean the working area and pipettes with an RNase decontamination solution. Use PCR clean and sterile dual-filter pipette tips and frequently change gloves to minimize contamination of reaction mixes with RNases.
3. Prepare the NTP/cap analog mixture as described in Table 3.
4. Mix the NTP/cap analog mixture thoroughly by vortexing and spin down briefly.
5. Assemble the IVT reaction mixture as described in Table 4.
6. Mix the IVT reaction mixture thoroughly by gently pipetting up and down. Then centrifuge the PCR tube briefly to collect the mixture at the bottom of the tube.
7. Incubate at 37 °C for 3 hr in a thermomixer.
   NOTE: The optimum incubation time depends on the length of the inserts and transcriptional efficiency of given template. For short inserts (<500 nt), a longer incubation time may be advantageous. A time-course experiment can be done to determine the optimum incubation time for maximum yield. Therefore, set up IVT reactions, for example, for 1, 2, 3, 4, 6 hr, and overnight incubation and determine the mRNA amount. The kinetics of the in vitro transcription for generation of eGFP mRNA is shown in Figure 3.
8. To remove the template DNA, add 1 µl of DNase (2 U/µl) to the IVT reaction mixture, mix well and incubate 15 min at 37 °C.
9. Purify the reaction mixture using a RNA purification kit according to manufacturer´s instructions. Elute the modified mRNA from the spin column membrane twice with 40 µl nuclease-free water. NOTE: The detected RNA concentration is approximately 1,500 µg/ml. Thus, the expected total amount should be ~120 µg. The amount of synthesized mRNA for other proteins can differ depending on the length of CDS and the amount of PCR product used for IVT.

5. Treatment of Purified mRNA with Antarctic Phosphatase

1. NOTE: The generated mRNA is treated with phosphatase to remove 5' triphosphates, which can be recognized by RIG-I and lead to immune activation. Furthermore, the phosphatase treatment prevents re-circularization in a self-ligation reaction.
2. Add 9 µl of 10x Antarctic phosphatase reaction buffer to 79 µl of purified mRNA solution. Subsequently, add 2 µl of Antarctic phosphatase (5 U/µl) and gently mix the sample. Incubate the reaction mixture at 37 °C for 30 min.
3. Purify the reaction mixture using a RNA purification kit according to manufacturer´s instructions. Elute the modified mRNA from the spin column membrane twice with 50 µl nuclease-free water.
4. Measure the concentration of the modified mRNA using a spectrophotometer. Check that the ratio of absorbance at 260 nm/280 nm (A₂₆₀/A₂₈₀) is ≥ 1.8 and the ratio of A₂₆₀/A₂₃₀ is 2.0, which indicates purity.
   NOTE: The expected total yield should be approximately 90 µg, which is sufficient for approximately 36 mRNA transfections.
5. Adjust the concentration of mRNA to 100 ng/µl by adding nuclease-free water.
6. Aliquot the modified mRNA into single-use aliquots required for transfections and store at -80 °C. Avoid multiple freeze and thaw cycles. Properly prepared and stored mRNA is stable for many years. During working, always keep the modified mRNA on ice.

6. Control Step: Quality of Synthesized Modified mRNA

1. Check the quality of modified mRNA by RNA gel electrophoresis.
2. Mix the desired amount of agarose with 1x TBE in a flask. For a 1% agarose gel, add 0.5 g of agarose to 50 ml of 1x TBE.
3. Microwave until the agarose is dissolved. Do not let it boil. Ensure that the agarose is completely in solution.
4. Add 5 µl nucleic acid gel stain to 50 ml agarose gel.
5. Pour the gel into a agarose gel box, set the comb, and wait approximately 1 hr until the gel is solidified.
6. Remove the comb and place the gel box in a gel tray.
7. Mix 3 µl (3 µg) of 0.5-10 kb RNA Ladder and 200 ng of synthesized modified mRNA each with 7 µl loading buffer in a total volume of 10 µl. The composition of the loading buffer is described in Table 5.
8. Denature the ladder and the mRNA samples at 70 °C for 10 min.
9. Carefully load the ladder and the mRNA samples on the 1% agarose gel.
10. Perform electrophoresis at 100 V for 1 hr using 1x TBE as running buffer.
11. Visualize the ladder and mRNA bands on a UV transilluminator and photograph using a gel documentation system (Figure 4).

7. Preparing of Cells for Transfection

1. Plate 2 x10⁵ cells (HEK293 cells) per well of 12-well plate.
2. Incubate the cells overnight at 37 °C in a cell incubator. NOTE: At the day of transfection, the cells should have reached 80-90% confluence.

8. Performing mRNA Transfection of Cells

1. Thaw the modified mRNA.
2. Generate the lipoplexes for transfection.
3. Add 25 µl (2.5 µg) of modified mRNA and 2 µl of cationic lipid transfection reagent to 473 µl Opti-MEM (Minimal Essential Medium) I reduced serum medium to generate the transfection mixture for transfection of one well of a 12-well plate. Scale up the volumes according to the number of wells to be transfected. As negative control, prepare a transfection mixture without mRNA.
4. Mix the components gently by pipetting. Incubate the transfection mixture at room temperature for 20 min to generate lipoplexes for transfection.
   NOTE: The transfection mixture contains no antibiotics. Thus, take care to ensure the sterility while handling cells.
5. Wash cells with 500 µl DPBS/well.
6. Add 500 µl transfection mixture to one well of a 12-well plate.
7. Incubate the cells for 4 hr at 37 °C and 5% CO₂.
8. Aspirate the transfection mixture and add 1 ml complete cell culture medium to the cells.
9. Incubate the cells for 24 hr in the cell incubator.
10. Perform fluorescence microscopic analyses using a fluorescence microscope (Figure 5). NOTE: To examine the expression of other proteins in cells, which are produced by transfection with other mRNA than eGFP coding mRNA, the cells can be cultivated on coverslips. Then the transfection can be performed and the cells can be stained with appropriate antibodies and analyzed by fluorescence microscopy.

9. Flow Cytometric Analyses of eGFP Expression in Cells

1. Remove cell culture medium from the wells and wash each well with 1 ml DPBS (without calcium and magnesium). Add 500 µl trypsin/EDTA per well to detach the cells from the surface of cell culture plates. Subsequently, add 500 µl TNS per well to inactivate trypsin.
2. Centrifuge the detached cells for 5 min at 300 x g at room temperature. Remove the supernatant leaving only the pellet. Resuspend the cell pellet in 150 µl cell fixation solution and transfer the cell suspension into a flow cytometry tube.
3. Analyze the percentage of eGFP expressing cells and the fluorescence intensity using Geo Mean (geometric mean of fluorescence intensity) (Figure 6).

10. Measurement of Protein Expression Over Time

1. Perform mRNA transfection of cells in 3 wells of a 12-well plate as described in step 8. Additionally, incubate 3 wells of HEK293 cells with the transfection mixture without the addition of mRNA.
2. Measure the expression of eGFP 1, 2, and 3 days after transfection to evaluate the duration of protein expression (Figure 7).

Results

Using a pcDNA 3.3 plasmid containing the CDS of eGFP, the synthesis of modified eGFP mRNA was established (Figure 1). After insert amplification by PCR and poly T-tailing, a clear band with a length of approximately 1,100 bp is detected (Figure 2). Increasing the IVT time augmented the yield of mRNA (Figure 3). After the IVT, a clear mRNA band with a length of approximately 1,100 bp was detected, which corresponds to the length of eGFP mRNA to be produced (Figure...

Discussion

The mRNA therapy has tremendous potential in the field of regenerative medicine, treatment of diseases and vaccination. In this video, we demonstrate the production of a stabilized, modified mRNA for induction of protein expression in cells. Using this protocol, other desired mRNA can be generated. The in vitro synthesis of modified mRNA allows the transfection of cells with desired mRNAs to induce expression of target proteins. Thereby, the desired protein is expressed transiently under physiological conditions...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cell culture
DMEM, high glucose	PAA	E15-009
FBS	Life Technologies	10500
Penicillin/Streptomycin	PAA	P11-010	100x
L-glutamine	PAA	M11-004	200 mM
DPBS without calcium and magnesium	PAA	E15-002
0.04% Trypsin / 0.03% EDTA	Promocell	C-41020
TNS	Promocell	C-41120	Trypsin Neutralizing Solution, 0.05% trypsin inhibitor in 0.1% BSA
HEK-293 cells	ATCC	CRL-1573
Consumables
Tissue culture plates, 12-well	Corning	3512
Cell culture flask (75 cm2)	Corning	430641
DNase-and RNase-free 1.5 ml sterile microcentrifuge tubes	Eppendorf	0030 121.589	Safe-Lock, Biopur
15 ml conical tubes	greiner bio-one	188271
PCR clean and sterile epT.I.P.S. dualfilter pipette tips	Eppendorf	10 µl: 022491202; 100 µl: 022491237; 1,000 µl: 022491253
Cryovial	greiner bio-one	122279-128	Cryo.s
14 ml polypropylene round bottom tube for bacterial culture	BD Falcon	352059
Plasmid amplification and purification
pcDNA 3.3_eGFP Plasmid	Addgene	26822
One Shot Top10 chemically component Escherichia coli	Invitrogen	C4040-10
Sterile water (Ampuwa)	Fresenius Kabi	1636071
LB medium (Luria/Miller)	Carl Roth	X968.1	Dissolve 25 g L-1 in sterile water.
LB agar (Luria/Miller)	Carl Roth	X969.1	Dissolve 40 g L-1 in sterile water.
Ampicillin Ready Made Solution	Sigma Aldrich	A5354	100 mg/ml
Glycerol	Sigma Aldrich	G2025
QIAprep Spin Miniprep Kit	Qiagen	27104
mRNA production
HotStar HiFidelity Polymerase Kit	Qiagen	202602
QIAquick PCR Purification Kit	Qiagen	28106
MEGAscript T7 Kit	Life Technologies	AM1334
5-Methylcytidine-5´-triphosphate	Trilink	N1014	5-Methyl-CTP
Pseudouridine-5´-triphosphate	Trilink	N1019	Pseudo-UTP
3´-O-Me-m7G(5´)ppp(5´)G RNA cap structure analog	New England Biolabs	S1411L
RiboLock RNase Inhibitor	Thermo Scientific	EO0381	40 U/µl
TURBO DNase	Life Technologies	AM1334	2 U/µl (from MEGAscript T7 Kit)
Antarctic phosphatase	New England Biolabs	MO289S
RNeasy mini kit	Qiagen	74104
RNaseZap solution	Life Technologies	AM9780
Transfection
Lipofectamine 2000 Transfection Reagent	Invitrogen	11668-019	Cationic lipid transfection reagent
Opti-MEM I Reduced Serum Media	Invitrogen	11058-021	Improved Minimal Essential Medium (MEM) that allows a reduction of Fetal Bovine Serum (FBS) supplementation
Gel electrophoresis
Agarose	Sigma-Aldrich	A9539
Gelred Nucleic Acid Gel Stain	Biotium	41003	10,000x in water
peqGOLD Range Mix DNA-Ladder	Peqlab	25-2210
Flow cytometry analyses
CellFIX (1x)	BD Biosciences	340181	10x concentrate
Primer for insert amplification and poly (T) tail PCR
Forward Primer (HPLC-grade) 10 µM	Ella Biotech		5´-TTGGACCCTCGTAC AGAAGCTAATACG-3´
Reverse Primer (HPLC-grade) 10 µM	Ella Biotech		5´- T120-CTTCCTACT CAGGCTTTATTCAA AGACCA-3´
Equipment
Cell incubator	Binder		CO2 (5%) and O2 (20%)
CASY cell counter	Schärfe System
Sterile workbench	BDK Luft-und Reinraumtecknik GmbH
Bacterial incubator	Incutec
Water bath
ScanDrop spectrophotometer	Analytic Jena
PCR thermocycler	Eppendorf
Microcentrifuge	Eppendorf
Vortex	peqlab
Thermomixer	Eppendorf
Gel apparatus for electrophoresis	Bio-Rad
Gel documentation system	Bio-Rad
FACScan System	BD Biosciences
Fluorescence microscope	Nikon
Phase-contrast microscope	Zeiss