This project was funded by the European Social Funds in Baden-Wuerttemberg, Germany.

1. Augmentation of Plasmids Containing Required Coding DNA Sequences (CDS)
<ol>
	<li>Pre-warm SOC medium, which is included in the transformation kit, to room temperature and the LB agar plates containing 100 &#181;g/ml ampicillin to 37 &#176;C. Equilibrate the water bath to 42 &#176;C.</li>
	<li>Thaw one vial of chemically component E. coli on ice.</li>
	<li>Add 1-5 &#181;l 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.</li>
	<li>Incubate the mixture on ice for 30 min.</li>
	<li>Heat-shock the E. coli for 30 sec at 42 &#176;C in a water bath without shaking.</li>
	<li>Place the vial on ice for 2 min.</li>
	<li>Add 250 &#181;l of pre-warmed SOC medium to the E. coli and shake the bacteria horizontally at 300 rpm for 1 hr at 37 &#176;C in a bacterial incubator.</li>
	<li>Spread 100 &#181;l and 150 &#181;l from transformation mix on pre-warmed LB agar plates, invert the plates and incubate overnight at 37 &#176;C.</li>
	<li>After colonies become visible, inoculate a single colony from each plate into a 15 ml culture tube containing 5 ml LB medium with 100 &#181;g/ml ampicillin. Incubate them overnight at 37 &#176;C with shaking at 300 rpm until culture is in late log or stationary phase.</li>
	<li>For long-term storage of transformed E. coli, pipet 75 &#181;l of 100% glycerol into the cryovial. Add 225 &#181;l of the bacterial culture (frozen stock will contain 25% glycerol), mix well and store the cryovial at -80 &#176;C.</li>
	<li>Isolate the plasmids containing the required CDS using a plasmid purification kit according to manufacturer&#39;s instructions.</li>
	<li>Determine the plasmid concentration using a spectrophotometer.</li>
	<li>Prepare aliquots of 20 &#181;l and store them at -20 &#176;C for prolonged time.</li>
</ol>
2. Amplification of Plasmid Inserts and Adding of Poly T-tail by Polymerase Chain Reaction (PCR)
<ol>
	<li>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.</li>
	<li>Prepare PCR mixture as detailed in Table 1.</li>
	<li>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 &#181;l PCR mixture can be used to obtain a sufficient yield of PCR product.</li>
	<li>Place the PCR tubes in a thermocycler and run the PCR using the PCR cycling protocol (Table 2).</li>
	<li>Clean up the PCR reaction using a PCR purification kit according to manufacturer&#39;s instructions and elute the DNA using 20 &#181;l nuclease-free water.</li>
	<li>Measure the concentration of the DNA using a spectrophotometer. NOTE: The detected DNA concentration is approximately 300 &#181;g/ml. Thus, the expected total amount should be ~6 &#181;g. 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.</li>
	<li>Freeze the DNA at -20 &#176;C for a long time or use it directly for IVT.</li>
</ol>
3. Control Step: Quality of PCR Product
<ol>
	<li>Check the quality of the PCR product by DNA gel electrophoresis.</li>
	<li>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.</li>
	<li>Microwave until the agarose is dissolved. Do not let it boil. Ensure that the agarose is completely in solution.</li>
	<li>Add 5 &#181;l nucleic acid gel stain to 50 ml agarose gel.</li>
	<li>Pour the gel into an agarose gel box, set the comb, and wait approximately 1 hr until the gel solidified.</li>
	<li>Remove the comb and place the gel box in a gel tray.</li>
	<li>Mix 2 &#181;l (200 ng) of 80-10,000 bp DNA-Ladder and 200 ng of PCR product each with 2 &#181;l 6x loading buffer in a total volume of 12 &#181;l.</li>
	<li>Carefully load DNA samples into the wells of the agarose gel.</li>
	<li>Using 1x TBE as running buffer, run the agarose gel at 100 V for 1 hr.</li>
	<li>Visualize the DNA bands on an imaging system (Figure 2).</li>
</ol>
4. In Vitro Transcription (IVT)
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Prepare the NTP/cap analog mixture as described in Table 3.</li>
	<li>Mix the NTP/cap analog mixture thoroughly by vortexing and spin down briefly.</li>
	<li>Assemble the IVT reaction mixture as described in Table 4.</li>
	<li>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.</li>
	<li>Incubate at 37 &#176;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 (&#60;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.</li>
	<li>To remove the template DNA, add 1 &#181;l of DNase (2 U/&#181;l) to the IVT reaction mixture, mix well and incubate 15 min at 37 &#176;C.</li>
	<li>Purify the reaction mixture using a RNA purification kit according to manufacturer&#180;s instructions. Elute the modified mRNA from the spin column membrane twice with 40 &#181;l nuclease-free water. NOTE: The detected RNA concentration is approximately 1,500 &#181;g/ml. Thus, the expected total amount should be ~120 &#181;g. 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.</li>
</ol>
5. Treatment of Purified mRNA with Antarctic Phosphatase
<ol>
	<li>NOTE: The generated mRNA is treated with phosphatase to remove 5&#39; 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.</li>
	<li>Add 9 &#181;l of 10x Antarctic phosphatase reaction buffer to 79 &#181;l of purified mRNA solution. Subsequently, add 2 &#181;l of Antarctic phosphatase (5 U/&#181;l) and gently mix the sample. Incubate the reaction mixture at 37 &#176;C for 30 min.</li>
	<li>Purify the reaction mixture using a RNA purification kit according to manufacturer&#180;s instructions. Elute the modified mRNA from the spin column membrane twice with 50 &#181;l nuclease-free water.</li>
	<li>Measure the concentration of the modified mRNA using a spectrophotometer. Check that the ratio of absorbance at 260 nm/280 nm (A260/A280) is &#8805; 1.8 and the ratio of A260/A230 is 2.0, which indicates purity. 
	NOTE: The expected total yield should be approximately 90 &#181;g, which is sufficient for approximately 36 mRNA transfections.</li>
	<li>Adjust the concentration of mRNA to 100 ng/&#181;l by adding nuclease-free water.</li>
	<li>Aliquot the modified mRNA into single-use aliquots required for transfections and store at -80 &#176;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.</li>
</ol>
6. Control Step: Quality of Synthesized Modified mRNA
<ol>
	<li>Check the quality of modified mRNA by RNA gel electrophoresis.</li>
	<li>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.</li>
	<li>Microwave until the agarose is dissolved. Do not let it boil. Ensure that the agarose is completely in solution.</li>
	<li>Add 5 &#181;l nucleic acid gel stain to 50 ml agarose gel.</li>
	<li>Pour the gel into a agarose gel box, set the comb, and wait approximately 1 hr until the gel is solidified.</li>
	<li>Remove the comb and place the gel box in a gel tray.</li>
	<li>Mix 3 &#181;l (3 &#181;g) of 0.5-10 kb RNA Ladder and 200 ng of synthesized modified mRNA each with 7 &#181;l loading buffer in a total volume of 10 &#181;l. The composition of the loading buffer is described in Table 5.</li>
	<li>Denature the ladder and the mRNA samples at 70 &#176;C for 10 min.</li>
	<li>Carefully load the ladder and the mRNA samples on the 1% agarose gel.</li>
	<li>Perform electrophoresis at 100 V for 1 hr using 1x TBE as running buffer.</li>
	<li>Visualize the ladder and mRNA bands on a UV transilluminator and photograph using a gel documentation system (Figure 4).</li>
</ol>
7. Preparing of Cells for Transfection
<ol>
	<li>Plate 2 x105 cells (HEK293 cells) per well of 12-well plate.</li>
	<li>Incubate the cells overnight at 37 &#176;C in a cell incubator. NOTE: At the day of transfection, the cells should have reached 80-90% confluence.</li>
</ol>
8. Performing mRNA Transfection of Cells
<ol>
	<li>Thaw the modified mRNA.</li>
	<li>Generate the lipoplexes for transfection.</li>
	<li>Add 25 &#181;l (2.5 &#181;g) of modified mRNA and 2 &#181;l of cationic lipid transfection reagent to 473 &#181;l 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.</li>
	<li>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.</li>
	<li>Wash cells with 500 &#181;l DPBS/well.</li>
	<li>Add 500 &#181;l transfection mixture to one well of a 12-well plate.</li>
	<li>Incubate the cells for 4 hr at 37 &#176;C and 5% CO2.</li>
	<li>Aspirate the transfection mixture and add 1 ml complete cell culture medium to the cells.</li>
	<li>Incubate the cells for 24 hr in the cell incubator.</li>
	<li>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.</li>
</ol>
9. Flow Cytometric Analyses of eGFP Expression in Cells
<ol>
	<li>Remove cell culture medium from the wells and wash each well with 1 ml DPBS (without calcium and magnesium). Add 500 &#181;l trypsin/EDTA per well to detach the cells from the surface of cell culture plates. Subsequently, add 500 &#181;l TNS per well to inactivate trypsin.</li>
	<li>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 &#181;l cell fixation solution and transfer the cell suspension into a flow cytometry tube.</li>
	<li>Analyze the percentage of eGFP expressing cells and the fluorescence intensity using Geo Mean (geometric mean of fluorescence intensity) (Figure 6).</li>
</ol>
10. Measurement of Protein Expression Over Time
<ol>
	<li>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.</li>
	<li>Measure the expression of eGFP 1, 2, and 3 days after transfection to evaluate the duration of protein expression (Figure 7).</li>
</ol>

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The authors declare that they have no competing financial interests.

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...

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.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Cell culture&lt;/strong&gt;</td></tr><tr><td>DMEM, high glucose</td><td>PAA</td><td>E15-009</td><td></td></tr><tr><td>FBS</td><td>Life Technologies</td><td>10500</td><td></td></tr><tr><td>Penicillin/Streptomycin&amp;nbsp;</td><td>PAA</td><td>P11-010</td><td>100x</td></tr><tr><td>L-glutamine&amp;nbsp;</td><td>PAA</td><td>M11-004</td><td>200 mM</td></tr><tr><td>DPBS without calcium and magnesium&amp;nbsp;</td><td>PAA</td><td>E15-002</td><td></td></tr><tr><td>0.04% Trypsin / 0.03% EDTA&amp;nbsp;</td><td>Promocell</td><td>C-41020</td><td></td></tr><tr><td>TNS</td><td>Promocell</td><td>C-41120</td><td>Trypsin Neutralizing Solution, 0.05% trypsin inhibitor in 0.1% BSA</td></tr><tr><td>HEK-293 cells</td><td>ATCC</td><td>CRL-1573</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Consumables&lt;/strong&gt;</td></tr><tr><td>Tissue culture plates, 12-well&amp;nbsp;</td><td>Corning</td><td>3512</td><td></td></tr><tr><td>Cell culture flask (75 cm&lt;sup&gt;2&lt;/sup&gt;)&amp;nbsp;</td><td>Corning</td><td>430641</td><td></td></tr><tr><td>DNase-and RNase-free 1.5 ml sterile microcentrifuge tubes&amp;nbsp;</td><td>Eppendorf</td><td>0030 121.589</td><td>Safe-Lock, Biopur</td></tr><tr><td>15 ml conical tubes&amp;nbsp;</td><td>greiner bio-one</td><td>188271</td><td></td></tr><tr><td>PCR clean and sterile epT.I.P.S. dualfilter pipette tips&amp;nbsp;</td><td>Eppendorf</td><td>10 &amp;micro;l: 022491202; 100 &amp;micro;l: 022491237; 1,000 &amp;micro;l: 022491253</td><td></td></tr><tr><td>Cryovial</td><td>greiner bio-one</td><td>122279-128</td><td>Cryo.s&amp;nbsp;</td></tr><tr><td>14 ml polypropylene round bottom tube for bacterial culture&amp;nbsp;</td><td>BD Falcon</td><td>352059</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Plasmid amplification and purification&lt;/strong&gt;</td></tr><tr><td>pcDNA 3.3_eGFP Plasmid&amp;nbsp;</td><td>Addgene</td><td>26822</td><td></td></tr><tr><td>One Shot Top10 chemically component Escherichia coli&amp;nbsp;</td><td>Invitrogen</td><td>C4040-10</td><td></td></tr><tr><td>Sterile water (Ampuwa)</td><td>Fresenius Kabi</td><td>1636071</td><td></td></tr><tr><td>LB medium (Luria/Miller)&amp;nbsp;</td><td>Carl Roth</td><td>X968.1</td><td>Dissolve 25 g L&lt;sup&gt;-1 &lt;/sup&gt;in sterile water.</td></tr><tr><td>LB agar (Luria/Miller)&amp;nbsp;</td><td>Carl Roth</td><td>X969.1</td><td>Dissolve 40 g L&lt;sup&gt;-1 &lt;/sup&gt;in sterile water.</td></tr><tr><td>Ampicillin Ready Made Solution</td><td>Sigma Aldrich</td><td>A5354</td><td>100 mg/ml&amp;nbsp;</td></tr><tr><td>Glycerol</td><td>Sigma Aldrich</td><td>G2025</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit&amp;nbsp;</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;mRNA production&lt;/strong&gt;</td></tr><tr><td>HotStar HiFidelity Polymerase Kit&amp;nbsp;</td><td>Qiagen</td><td>202602</td><td></td></tr><tr><td>QIAquick PCR Purification Kit&amp;nbsp;</td><td>Qiagen</td><td>28106</td><td></td></tr><tr><td>MEGAscript T7 Kit&amp;nbsp;</td><td>Life Technologies</td><td>AM1334</td><td></td></tr><tr><td>5-Methylcytidine-5&amp;acute;-triphosphate&amp;nbsp;</td><td>Trilink</td><td>N1014</td><td>5-Methyl-CTP</td></tr><tr><td>Pseudouridine-5&amp;acute;-triphosphate&amp;nbsp;</td><td>Trilink</td><td>N1019</td><td>Pseudo-UTP</td></tr><tr><td>3&amp;acute;-&lt;em&gt;O&lt;/em&gt;-Me-m&lt;sup&gt;7&lt;/sup&gt;G(5&amp;acute;)ppp(5&amp;acute;)G RNA cap structure analog&amp;nbsp;</td><td>New England Biolabs</td><td>S1411L</td><td></td></tr><tr><td>RiboLock RNase Inhibitor</td><td>Thermo Scientific</td><td>EO0381</td><td>40 U/&amp;micro;l&amp;nbsp;</td></tr><tr><td>TURBO DNase</td><td>Life Technologies</td><td>AM1334</td><td>2 U/&amp;micro;l (from MEGAscript T7 Kit)</td></tr><tr><td>Antarctic phosphatase&amp;nbsp;</td><td>New England Biolabs</td><td>MO289S</td><td></td></tr><tr><td>RNeasy mini kit&amp;nbsp;</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>RNaseZap solution&amp;nbsp;</td><td>Life Technologies</td><td>AM9780</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Transfection&lt;/strong&gt;</td></tr><tr><td>Lipofectamine 2000 Transfection Reagent&amp;nbsp;</td><td>Invitrogen</td><td>11668-019</td><td>Cationic lipid transfection reagent</td></tr><tr><td>Opti-MEM I Reduced Serum Media&amp;nbsp;</td><td>Invitrogen</td><td>11058-021</td><td>Improved Minimal Essential Medium (MEM) that allows a reduction of Fetal Bovine Serum (FBS) supplementation&amp;nbsp;</td></tr><tr><td colspan="4">&lt;strong&gt;Gel electrophoresis&lt;/strong&gt;</td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A9539</td><td></td></tr><tr><td>Gelred Nucleic Acid Gel Stain</td><td>Biotium</td><td>41003</td><td>10,000x in water&amp;nbsp;</td></tr><tr><td>peqGOLD Range Mix DNA-Ladder&amp;nbsp;</td><td>Peqlab</td><td>25-2210</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Flow cytometry analyses&lt;/strong&gt;</td></tr><tr><td>CellFIX (1x)</td><td>BD Biosciences</td><td>340181</td><td>10x concentrate&amp;nbsp;</td></tr><tr><td colspan="4">&lt;strong&gt;Primer for insert amplification and poly (T) tail PCR&lt;/strong&gt;</td></tr><tr><td>Forward Primer (HPLC-grade) 10 &amp;micro;M</td><td>Ella Biotech</td><td></td><td>5&amp;acute;-TTGGACCCTCGTAC&lt;br /&gt;AGAAGCTAATACG-3&amp;acute;</td></tr><tr><td>Reverse Primer (HPLC-grade) 10 &amp;micro;M</td><td>Ella Biotech</td><td></td><td>5&amp;acute;- T&lt;sub&gt;120&lt;/sub&gt;-CTTCCTACT&lt;br /&gt;CAGGCTTTATTCAA&lt;br /&gt;AGACCA-3&amp;acute;</td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Cell incubator</td><td>Binder</td><td></td><td>CO&lt;sub&gt;2&lt;/sub&gt; (5%) and O&lt;sub&gt;2&lt;/sub&gt; (20%)&amp;nbsp;</td></tr><tr><td>CASY cell counter&amp;nbsp;</td><td>Sch&amp;auml;rfe System</td><td></td><td></td></tr><tr><td>Sterile workbench&amp;nbsp;</td><td>BDK Luft-und Reinraumtecknik GmbH</td><td></td><td></td></tr><tr><td>Bacterial incubator&amp;nbsp;</td><td>Incutec</td><td></td><td></td></tr><tr><td>Water bath</td><td></td><td></td><td></td></tr><tr><td>ScanDrop spectrophotometer&amp;nbsp;</td><td>Analytic Jena</td><td></td><td></td></tr><tr><td>PCR thermocycler&amp;nbsp;</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Vortex</td><td>peqlab</td><td></td><td></td></tr><tr><td>Thermomixer</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Gel apparatus for electrophoresis&amp;nbsp;</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Gel documentation system&amp;nbsp;</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>FACScan System&amp;nbsp;</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Fluorescence microscope&amp;nbsp;</td><td>Nikon</td><td></td><td></td></tr><tr><td>Phase-contrast microscope&amp;nbsp;</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

in vitro synthesis of modified mrna for induction of protein expression in human cells

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.

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...

Watch this Scientific Journal Video about In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells at JoVE.com

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

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

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

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

in-vitro-synthesis-modified-mrna-for-induction-protein-expression

Research

JoVE Journal

Biology

24.9K Views.  University Hospital Tuebingen. In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in cells.

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

Cell Based Assays of SINEUP Non-coding RNAs That Can Specifically Enhance mRNA Translation

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. Here, we present a method to selectively up-regulate protein expression of desired genes in cultured cells by means of synthetic antisense non-coding RNAs known as SINEUPs. This positive control of gene expression is at the post-transcriptional level and exerted by an inverted short interspersed nuclear element (SINE) repeat at the 3&#697; end of SINEUPs that comprises its effector domain (ED). SINEUPs can specifically bind to any protein-coding mRNA of choice through its binding domain (BD), a region designed to complement the sequence within the 5&#697; untranslated region (5&#697; UTR) and around the start codon of the mRNA. Target-specific SINEUPs designed in this manner are transfected to cultured cells, and protein and RNA are extracted for downstream analyses, generally 24-48 h post-transfection. SINEUP-induced protein up-regulation is detected by Western-blot analysis and RNA expression is measured using real-time quantitative reverse transcription PCR. We have observed that BD design is critical for achieving optimum SINEUP activity and that testing different BD sizes and positions with respect to the start codon of the target mRNA is recommended. Therefore, we describe here a semi-automated high-throughput imaging method based on fluorescence detection that can be implemented to target mRNA fused with green fluorescent protein (GFP). SINEUPs specifically enhance translation within normal physiological range of the cell, without altering the target transcript level. This method has been successfully employed against a range of endogenous and exogenous targets, in a wide variety of human, mouse, and insect cell lines along with in vivo systems. Moreover, SINEUPs have been reported to increase antibody production and work as an RNA therapeutic against haploinsufficient genes. The versatile and modular nature of SINEUPs makes them a suitable tool for gene-specific translational control.

SINEUPs are synthetic antisense non-coding RNAs, which contain a binding domain (BD) and an effector domain (ED) and up-regulate translation of target mRNA. Here, we describe detection methods for SINEUPs in cultured cell lines, analysis of their translation-promoting activity by Western-blot and a semi-automated high throughput imaging system.

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. ...

Genetics

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Developmental Biology

Using Lipid Nanoparticles for the Delivery of Chemically Modified mRNA into Mammalian Cells

In recent years, chemically modified messenger RNA (mRNA) has emerged as a potent nucleic acid molecule for developing a wide range of therapeutic applications, including a novel class of vaccines, protein replacement therapies, and immune therapies. Among delivery vectors, lipid nanoparticles are found to be safer and more effective in delivering RNA molecules (e.g., siRNA, miRNA, mRNA) and a few products are already in clinical use. To demonstrate lipid nanoparticle-mediated mRNA delivery, we present an optimized protocol for the synthesis of functional me1&#936;-UTP modified eGFP mRNA, the preparation of cationic liposomes, the electrostatic complex formation of mRNA with cationic liposomes, and the evaluation of transfection efficiencies in mammalian cells. The results demonstrate that these modifications efficiently improved the stability of mRNA when delivered with cationic liposomes and increased the eGFP mRNA translation efficiency and stability in mammalian cells. This protocol can be used to synthesize the desired mRNA and transfect with cationic liposomes for target gene expression in mammalian cells.

The protocol presents in vitro transcription (IVT) of chemically modified mRNA, cationic liposome preparation, and functional analysis of liposome enabled mRNA transfections in mammalian cells.

In recent years, chemically modified messenger RNA (mRNA) has emerged as a potent nucleic acid molecule for developing a wide range of therapeutic ...

Biochemistry

CFPS Protocols for Micro-Compartmentalized Synthetic Cells

Cell-Free Protein Synthesis System for Building Synthetic Cells

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for the core machinery of the Central Dogma, standing as an optimal chassis for the integration and assembly of diverse artificial cellular mimicry systems. Despite its frequent use in the fabrication of synthetic cells, establishing a tailored and robust CFPS system for a specific application remains a nontrivial challenge. In this methods paper, we present a comprehensive protocol for the CFPS system, routinely employed in constructing synthetic cells. This protocol encompasses key stages in the preparation of the CFPS system, including the cell extract, template preparation, and routine expression optimization utilizing a fluorescent reporter protein. Additionally, we show representative results by encapsulating the CFPS system within various micro-compartments, such as monolayer droplets, double-emulsion vesicles, and chambers situated atop supported lipid bilayers. Finally, we elucidate the critical steps and conditions necessary for the successful assembly of these CFPS systems in distinct environments. We expect that our approach will facilitate the establishment of good working practices among various laboratories within the continuously expanding synthetic cell community, thereby accelerating progress in the field of synthetic cell development.

Author Spotlight: Optimizing CFPS Systems for Synthetic Cell Construction

This protocol describes the Cell-Free Protein Synthesis (CFPS) system used in constructing synthetic cells. It outlines key stages with representative results in different micro-compartments. The protocol aims to establish best practices for diverse laboratories in the synthetic cell community, advancing progress in synthetic cell development.

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for ...

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive properties of in vitro transcribed (IVT) mRNA. With the help of IVT mRNA, the de novo synthesis of a desired protein can be induced without changing the physiological state of the target cell. Moreover, protein biosynthesis can be precisely controlled due to the transient effect of IVT mRNA.
For the efficient transfection of cells, nanoliposomes (NLps) may represent a safe and efficient delivery vehicle for therapeutic mRNA. This study describes a protocol to generate safe and efficient cationic NLps consisting of DC-cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a delivery vector for IVT mRNA. NLps having a defined size, a homogeneous distribution, and a high complexation capacity, and can be produced using the dry-film method. Moreover, we present different test systems to analyze their complexation and transfection efficacies using synthetic enhanced green fluorescent protein (eGFP) mRNA, as well as their effect on cell viability. Overall, the presented protocol provides an effective and safe approach for mRNA complexation, which may advance and improve the administration of therapeutic mRNA.

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive ...

Bioengineering

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...

Immunology and Infection

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

In vitro transcribed messenger RNA (mRNA) vaccines have displayed enormous potential in fighting against the coronavirus disease 2019 (COVID-19) pandemic. Efficient and safe delivery systems must be included in the mRNA vaccines due to the fragile properties of mRNA. A self-assembled peptide-poloxamine nanoparticle (PP-sNp) gene delivery system is specifically designed for the pulmonary delivery of nucleic acids and displays promising capabilities in mediating successful mRNA transfection. Here, an improved method for preparing PP-sNp is described to elaborate on how the PP-sNp&#160;encapsulates Metridia luciferase (MetLuc) mRNA and successfully transfects cultured cells. MetLuc-mRNA is obtained by an in vitro transcription process from a linear DNA template. A PP-sNp&#160;is produced by mixing synthetic peptide/poloxamine with mRNA solution using a microfluidic mixer, allowing for the self-assembly of PP-sNp. The charge of PP-sNp&#160;is subsequently evaluated by measuring the zeta potential. Meanwhile, the polydispersity and hydrodynamic size of PP-sNp&#160;nanoparticles are measured using dynamic light scattering. The mRNA/PP-sNp nanoparticles are transfected into cultured cells, and supernatants from the cell culture are assayed for luciferase activity. The representative results demonstrate their capacity for in vitro transfection. This protocol may shed light on developing next-generation mRNA vaccine delivery systems.

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

A self-assembled peptide-poloxamine nanoparticle (PP-sNp) is developed using a microfluidic mixing device to encapsulate and deliver in vitro transcribed messenger RNA. The described mRNA/PP-sNp could efficiently transfect cultured cells in vitro.

In vitro transcribed messenger RNA (mRNA) vaccines have displayed enormous potential in fighting against the coronavirus disease 2019 (COVID-19) pandemic. ...

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

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

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Rozdziały w tym wideo

Cell Based Assays of SINEUP Non-coding RNAs That Can Specifically Enhance mRNA Translation

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Using Lipid Nanoparticles for the Delivery of Chemically Modified mRNA into Mammalian Cells

Cell-Free Protein Synthesis System for Building Synthetic Cells

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension