Name: in vitro synthesis of modified mrna for induction of protein expression in human cells
Uploaded: 2014-11-13T13:00:00
Description: Watch this Scientific Journal Video about In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells at JoVE.com

Dit project werd gefinancierd door het Europees Sociaal Fonds in Baden-Wuerttemberg, Duitsland.

1. Vergroting van plasmiden die Vereiste codering van DNA-sequenties (CDS) <ol><li> Voorverwarmen SOC medium, dat is opgenomen in de transformatie kit, tot kamertemperatuur en de LB-agarplaten die 100 ug / ml ampicilline bij 37 ° C. Equilibreer het waterbad tot 42 ° C. </li><li> Dooi een flacon van chemisch component E. coli op ijs. </li><li> Voeg 1-5 ul van het plasmide (10 pg tot 100 ng) met de CDS in de flacon met chemisch component E. coli en meng voorzichtig. Laat de cellen niet meng door pipe...

<ol>
	<li>Bangel-Ruland, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CFTR-mRNA+delivery:+a+novel+alternative+for+cystic+fibrosis+&amp;#34;gene+therapy&amp;#34;.">CFTR-mRNA delivery: a novel alternative for cystic fibrosis &amp;#34;gene therapy&amp;#34;.</a> The journal of gene medicine. , (2013).</li><li>Benteyn, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+an+Optimized+Wilms&amp;#34;+Tumor+1+(WT1)+mRNA+Construct+for+Enhanced+WT1+Expression+and+Improved+Immunogenicity+In+Vitro+and+In+Vivo.">Design of an Optimized Wilms&amp;#34; Tumor 1 (WT1) mRNA Construct for Enhanced WT1 Expression and Improved Immunogenicity In Vitro and In Vivo.</a> Molecular therapy Nucleic acids. 2, 134 (2013).</li><li>Petsch, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+efficacy+of+in+vitro+synthesized,+specific+mRNA+vaccines+against+influenza+A+virus+infection.">Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection.</a> Nature. 30, 1210-1216 (2012).</li><li>Mandal, P. K., Rossi, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+human+fibroblasts+to+pluripotency+using+modified+mRNA.">Reprogramming human fibroblasts to pluripotency using modified mRNA.</a> Nature protocols. 8, 568-582 (2013).</li><li>Warren, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+reprogramming+to+pluripotency+and+directed+differentiation+of+human+cells+with+synthetic+modified+mRNA.">Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.</a> Cell stem cell. 7, 618-630 (2010).</li><li>Yakubov, E., Rechavi, G., Rozenblatt, S., Givol, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+human+fibroblasts+to+pluripotent+stem+cells+using+mRNA+of+four+transcription+factors.">Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors.</a> Biochemical and biophysical research communications. 394, 189-193 (2010).</li><li>Anderson, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+pseudouridine+into+mRNA+enhances+translation+by+diminishing+PKR+activation.">Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation.</a> Nucleic acids research. 38, 5884-5892 (2010).</li><li>Kariko, K., Buckstein, M., Ni, H., Weissman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+RNA+recognition+by+Toll-like+receptors:+the+impact+of+nucleoside+modification+and+the+evolutionary+origin+of+RNA.">Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA.</a> Immunity. 23, 165-175 (2005).</li><li>Kariko, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+pseudouridine+into+mRNA+yields+superior+nonimmunogenic+vector+with+increased+translational+capacity+and+biological+stability.">Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability.</a> Molecular therapy : the journal of the American Society of Gene Therapy. 16, 1833-1840 (2008).</li><li>Kariko, K., Weissman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+occurring+nucleoside+modifications+suppress+the+immunostimulatory+activity+of+RNA:+implication+for+therapeutic+RNA+development.">Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development.</a> Current opinion in drug discovery &amp; development. 10, 523-532 (2007).</li><li>Kormann, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+therapeutic+proteins+after+delivery+of+chemically+modified+mRNA+in+mice.">Expression of therapeutic proteins after delivery of chemically modified mRNA in mice.</a> Nature biotechnology. 29, 154-157 (2011).</li><li>Hacein-Bey-Abina, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertional+oncogenesis+in+4+patients+after+retrovirus-mediated+gene+therapy+of+SCID-X1.">Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1.</a> The Journal of clinical investigation. 118, 3132-3142 (2008).</li><li>Hacein-Bey-Abina, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+gene+therapy+for+X-linked+severe+combined+immunodeficiency.">Efficacy of gene therapy for X-linked severe combined immunodeficiency.</a> The New England journal of medicine. 363, 355-364 (2010).</li><li>Avci-Adali, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+conditions+for+successful+transfection+of+human+endothelial+cells+with+in+vitro+synthesized+and+modified+mRNA+for+induction+of+protein+expression.">Optimized conditions for successful transfection of human endothelial cells with in vitro synthesized and modified mRNA for induction of protein expression.</a> Journal of biological engineering. 8, 8 (2014).</li></ol>

De mRNA therapie enorm potentieel op het gebied van regeneratieve geneeskunde, behandeling van ziekten en vaccinatie. In deze video tonen we de productie van een gestabiliseerd, gemodificeerd mRNA voor inductie van eiwitexpressie in cellen. Met dit protocol kunnen andere gewenste mRNA gegenereerd. De in vitro synthese van gemodificeerde mRNA kan de transfectie van cellen met gewenste mRNA expressie van doeleiwitten induceren. Daardoor wordt het gewenste eiwit transiënt tot expressie gebracht onder fysiologisch...

In cellen de transcriptie van messenger RNA (mRNA) en de volgende translatie van mRNA in de gewenste eiwitten de goede werking van de cellen. Erfelijke of verworven genetische aandoeningen kan leiden tot onvoldoende en disfunctionele synthese van eiwitten en ernstige ziekten. Dus een nieuwe therapeutische benadering voor de produktie van ontbrekende of defecte eiwitten induceren exogene levering van synthetisch gemodificeerde mRNA in cellen, die codeert voor het gewenste eiwit. Daardoor worden cellen geactiveerd om functionele eiwitten, die ze normaal niet produceert of zou uiteraard niet moeten synthetiseren. Met deze benadering kunnen genetische aandoeningen worden gecorrigeerd door het inbrengen van mRNA dat codeert voor de defecte of ontbrekende eiwit 1. De mRNA therapie kan ook worden gebruikt voor vaccinatie eiwit antigenen die worden uitgedrukt door tumorcellen of pathogenen synthetiseren. Daardoor kan gastheerimmuunsysteem worden geactiveerd om effectief elimineren tumorcellen of Infe voorkomenCTIES 2,3. Bovendien afgelopen jaren mRNA succes gebruikt geïnduceerde pluripotente stamcellen (iPSCs) genereren. Hiertoe werden fibroblasten getransfecteerd met mRNA induceren de expressie van herprogrammering factoren 4-6 en om te zetten in iPSCs met een enorm potentieel regeneratieve geneeskunde. Eerder is het gebruik van conventionele mRNA geassocieerd met lage stabiliteit en sterke immunogeniciteit. Aldus klinische toepassingen van conventionele mRNA beperkt. De vervanging van cytidine en uridine van 5-methylcytidine en pseudouridine in het mRNA-molecuul door Kariko en collega rendered mRNA-moleculen stabiel in biologische vloeistoffen en drastisch verminderd immuunactivatie 7-10, die nu kan de klinische toepasbaarheid van gemodificeerde mRNA. Met synthetisch geproduceerde gemodificeerde mRNA, kan gewenst gen transcripten tijdelijk worden geleverd in vivo 11of in vitro om eiwitexpressie te induceren. De geïntroduceerde mRNA wordt vertaald onder fysiologische omstandigheden door de cellulaire translatie machinerie. Wegens gebrek aan integratie in het gastheergenoom opzichte virale gentherapie vectoren, is het risico van oncogenese voorkomen 12,13. Zo zal de behandeling met behulp van gemodificeerde synthetische mRNA krijgen betere klinische acceptatie in de toekomst. Hier beschrijven we een gedetailleerd protocol voor de productie van gemodificeerde mRNA (figuur 1), transfectie van cellen met mRNA en de evaluatie van eiwitexpressie in getransfecteerde cellen.

<table border="0" cellpadding="0" cellspacing="0" width="1058">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Name of Material/ Equipment</td>
			<td style="width:157px;">Company</td>
			<td style="width:172px;">Catalog Number</td>
			<td style="width:435px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Cell culture</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, high glucose</td>
			<td>PAA</td>
			<td>E15-009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Life Technologies</td>
			<td>10500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin&#160;</td>
			<td style="width:157px;">PAA</td>
			<td>P11-010</td>
			<td>100x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine&#160;</td>
			<td>PAA</td>
			<td>M11-004</td>
			<td>200 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS without calcium and magnesium&#160;</td>
			<td>PAA</td>
			<td>E15-002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.04% Trypsin / 0.03% EDTA&#160;</td>
			<td>Promocell</td>
			<td>C-41020</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">TNS</td>
			<td>Promocell</td>
			<td>C-41120</td>
			<td style="width:435px;">Trypsin Neutralizing Solution, 0.05% trypsin inhibitor in 0.1% BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEK-293 cells</td>
			<td style="width:157px;">ATCC</td>
			<td>CRL-1573</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Consumables</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture plates, 12-well&#160;</td>
			<td>Corning</td>
			<td>3512</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Cell culture flask (75 cm2)&#160;</td>
			<td>Corning</td>
			<td>430641</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">DNase-and RNase-free 1.5 ml sterile microcentrifuge tubes&#160;</td>
			<td>Eppendorf</td>
			<td>0030 121.589</td>
			<td>Safe-Lock, Biopur</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 ml conical tubes&#160;</td>
			<td>greiner bio-one</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:295px;">PCR clean and sterile epT.I.P.S. dualfilter pipette tips&#160;</td>
			<td>Eppendorf</td>
			<td style="width:172px;">10 &#181;l: 022491202,&#160;&#160;&#160; 100 &#181;l: 022491237, 1000 &#181;l: 022491253</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryovial</td>
			<td>greiner bio-one</td>
			<td>122279-128</td>
			<td>Cryo.s&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">14 ml polypropylene round bottom tube for bacterial culture&#160;</td>
			<td>BD Falcon</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasmid amplification and purification</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pcDNA 3.3_eGFP Plasmid&#160;</td>
			<td style="width:157px;">Addgene</td>
			<td>26822</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">One Shot Top10 chemically component Escherichia coli&#160;</td>
			<td>Invitrogen</td>
			<td>C4040-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Sterile water (Ampuwa)</td>
			<td>Fresenius Kabi</td>
			<td>1636071</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">LB medium (Luria/Miller)&#160;</td>
			<td>Carl Roth</td>
			<td>X968.1</td>
			<td>Dissolve 25 g l-1 in sterile water.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">LB agar (Luria/Miller)&#160;</td>
			<td>Carl Roth</td>
			<td>X969.1</td>
			<td>Dissolve 40 g l-1 in sterile water.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin Ready Made Solution</td>
			<td>Sigma Aldrich</td>
			<td>A5354</td>
			<td>100 mg/ml&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Sigma Aldrich</td>
			<td>G2025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAprep Spin Miniprep Kit&#160;</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mRNA production</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HotStar HiFidelity Polymerase Kit&#160;</td>
			<td>Qiagen</td>
			<td>202602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick PCR Purification Kit&#160;</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEGAscript T7 Kit&#160;</td>
			<td>Life Technologies</td>
			<td>AM1334</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-Methylcytidine-5&#180;-triphosphate&#160;</td>
			<td>Trilink</td>
			<td>N1014</td>
			<td>5-Methyl-CTP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pseudouridine-5&#180;-triphosphate&#160;</td>
			<td>Trilink</td>
			<td>N1019</td>
			<td>Pseudo-UTP</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:295px;">3&#180;-O-Me-m7G(5&#180;)ppp(5`)G RNA cap structure analog&#160;</td>
			<td>New England Biolabs</td>
			<td>S1411L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RiboLock RNase Inhibitor</td>
			<td>Thermo Scientific</td>
			<td>EO0381</td>
			<td>40 U/&#181;l&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TURBO DNase</td>
			<td>Life Technologies</td>
			<td>AM1334</td>
			<td>2 U/&#181;l (from MEGAscript T7 Kit)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antarctic phosphatase&#160;</td>
			<td>New England Biolabs</td>
			<td>MO289S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNeasy mini kit&#160;</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNaseZap solution&#160;</td>
			<td>Life Technologies</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfection</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine 2000 Transfection Reagent&#160;</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td>Cationic lipid transfection reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Opti-MEM I Reduced Serum Media&#160;</td>
			<td>Invitrogen</td>
			<td>11058-021</td>
			<td style="width:435px;">Improved Minimal Essential Medium (MEM) with&#160; reduced Fetal Bovine Serum (FBS) supplementation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel electrophoresis</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelred Nucleic Acid Gel Stain</td>
			<td>Biotium</td>
			<td>41003</td>
			<td>10,000x in water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">peqGOLD Range Mix DNA-Ladder&#160;</td>
			<td>Peqlab</td>
			<td>25-2210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5-10 kb RNA Ladder&#160;</td>
			<td>Invitrogen</td>
			<td>15623-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1x TBE buffer</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow cytometry analyses</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellFIX (1x)</td>
			<td>BD Biosciences</td>
			<td>340181</td>
			<td>10x concentrate&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Primer for insert amplification and&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; poly (T) tail PCR</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forward Primer (HPLC-grade) 10 &#181;M</td>
			<td style="width:157px;">Ella Biotech</td>
			<td style="width:172px;"></td>
			<td>5&#180;-TTGGACCCTCGTACAGAAGCTAATACG-3&#180;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Reverse Primer (HPLC-grade) 10 &#181;M</td>
			<td style="width:157px;">Ella Biotech</td>
			<td style="width:172px;"></td>
			<td>5&#180;- T120-CTTCCTACTCAGGCTTTATTCAAAGACCA-3&#180;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Cell incubator</td>
			<td>Binder</td>
			<td style="width:172px;"></td>
			<td>CO2 (5%) and O2 (20%)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CASY cell counter&#160;</td>
			<td>Sch&#228;rfe System</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;">Sterile workbench&#160;</td>
			<td style="width:157px;">BDK Luft-und Reinraumtecknik GmbH</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacterial incubator&#160;</td>
			<td>Incutec</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water bath</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ScanDrop spectrophotometer&#160;</td>
			<td>Analytic Jena</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR thermocycler&#160;</td>
			<td>Eppendorf</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge</td>
			<td>Eppendorf</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex</td>
			<td>peqlab</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermomixer</td>
			<td>Eppendorf</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel apparatus for electrophoresis&#160;</td>
			<td>Bio-Rad</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel documentation system&#160;</td>
			<td>Bio-Rad</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACScan System&#160;</td>
			<td>BD Biosciences</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence microscope&#160;</td>
			<td>Nikon</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phase-contrast microscope&#160;</td>
			<td>Zeiss</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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

De afgifte van exogene coderende synthetische messenger RNA (mRNA) voor inductie van eiwitsynthese in gewenste cellen enorm potentieel op het gebied van regeneratieve geneeskunde, fundamentele celbiologie, behandeling van ziekten en herprogrammering van cellen. We beschrijven hier een stapsgewijze protocol voor het genereren van gemodificeerde mRNA met verminderde immuunactivatie en potentieel verhoogde stabiliteit, kwaliteitscontrole van geproduceerde mRNA, transfectie van cellen met mRNA en verificatie van de eiwitexpressie geïnduceerd door flowcytometrie. Tot 3 dagen na een transfectie met eGFP mRNA, de getransfecteerde HEK293 cellen eGFP. In deze video artikel, wordt de synthese van eGFP mRNA beschreven als een voorbeeld. Echter, de procedure worden toegepast voor de productie van andere gewenste mRNA. Met de synthetische gemodificeerde mRNA, kunnen cellen worden geïnduceerd om de gewenste eiwitten, die zij normaal gesproken niet tot expressie transient expressie.

Met een pcDNA 3.3 plasmide dat de CDS van eGFP, de synthese van gemodificeerde eGFP mRNA werd vastgesteld (figuur 1). Na insertie amplificatie met PCR en poly T-tailing is een duidelijke band met een lengte van ongeveer 1100 bp gedetecteerd (Figuur 2). Verhogen van de IVT tijd Augmented de opbrengst van mRNA (figuur 3). Na de IVT, werd een duidelijke band mRNA met een lengte van ongeveer 1100 bp gedetecteerd, hetgeen overeenkomt met de lengte van eGFP mRNA te produceren...

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 vitro Synthese van gemodificeerde mRNA voor inductie van eiwitexpressie in menselijke cellen

In deze video artikel beschrijven we de in vitro synthese van gemodificeerde mRNA voor inductie van eiwitexpressie in cellen.

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

Research

JoVE Journal

Biology

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

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

Kleuring van eiwitten in Gels met Coomassie G-250 zonder organisch oplosmiddel en azijnzuur

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAÃƒÅ½Ã¢â‚¬Â41

Zuivering van het M. magneticum Strain AMB-1 Magnetosome geassocieerd eiwit MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

In vitro Reconstitution of the Active T. castaneum Telomerase

In vitro Reconstitution of the Active T. castaneum Telomerase

In vitro reconstitutie van de Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

Efficiënte Afleiding van de Mens Cardiac precursoren en cardiomyocyten uit pluripotente menselijke embryonale stamcellen met op kleine moleculen Inductie

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Isolatie van Ribosoom Bound Nascent Polypeptiden<em&gt; In vitro</em&gt; Om Translationeel Pauze plaatsen langs mRNA identificeren

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

Efficiënte Generation humane geïnduceerde pluripotente stamcellen uit menselijke somatische cellen met Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Profilering Individuele menselijke embryonale stamcellen door kwantitatieve RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Analyse van Global RNA synthese in de Single Cell Level volgende Hypoxie

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

siRNA Screening naar Ubiquitin en Ubiquitin-achtig systeem regulatoren van biologische pathways identificeren in gekweekte zoogdiercellen

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein gebaseerd Expression Screening van membraaneiwitten in<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...