פרויקט זה מומן על ידי קרנות החברתיות האירופיות באדן-וירטמברג, גרמניה.

<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>

החוקרים מצהירים כי אין להם אינטרסים כלכליים מתחרים.

יש טיפול mRNA פוטנציאל עצום בתחום של רפואת רגנרטיבית, טיפול במחלות וחיסונים. בסרטון הזה, אנחנו מדגימים את הייצור של ה- mRNA התייצב, שונה לזירוז של ביטוי חלבון בתאים. שימוש בפרוטוקול זה, יכול להיות שנוצר mRNA הרצוי אחר. הסינתזה במבחנה של mRNA שונה מאפשרת transfection של תאים עם mRN...

בתאים, השעתוק של RNA שליח (mRNA) והתרגום הבא של ה- mRNA לחלבונים רצויים להבטיח את התפקוד התקין של תאים. הפרעות גנטיות תורשתיות או נרכשות יכולות להוביל לסינתזה מספיקה ולא מתפקדת של חלבונים וגורמות למחלות קשות. כך, גישה טיפולית חדשה ללעורר את הייצור של חלבונים חסרים או פגומים הוא המשלוח אקסוגני של סינטטי שונה mRNA לתאים, שקודים לחלבון הרצוי. וכך, תאים מופעלים לסנתז חלבונים פונקציונליים, שבו הם בדרך כלל לא יכולים לייצר או באופן טבעי לא צריכים. שימוש בגישה זו, ניתן לתקן הפרעות גנטיות על ידי הקדמה של mRNA שמקודד לחלבון הפגום או חסר 1. גם טיפול mRNA יכול לשמש לחיסון לאנטיגנים synthetize חלבון, אשר באים לידי ביטוי על ידי תאי גידול או פתוגנים. ובכך, מערכת חיסונית מארח יכולה להיות מופעלת כדי לחסל ביעילות תאים סרטניים או למנוע infections 2,3. יתר על כן, בשנים האחרונות, mRNA היה בהצלחה המשמש לייצור תאים מושרה pluripotent גזע (iPSCs). לצורך כך, fibroblasts היה transfected עם mRNAs לגרום הביטוי של תכנות מחדש גורמי 4-6 ולהמיר אותם בiPSCs עם פוטנציאל עצום ברפואת רגנרטיבית. בעבר, השימוש בmRNA הקונבנציונלי היה קשור ביציבות נמוכה וחיסוני חזקים. כך, יישומים קליניים של mRNAs הקונבנציונלי היו מוגבלים. עם זאת, החלפת cytidine וuridine על ידי 5-methylcytidine וpseudouridine בתוך מולקולת mRNA על ידי Kariko ועמיתים שניתנו מולקולות mRNA יציבים בנוזלים ביולוגיים והפחתה דרמטית בהפעלת מערכת חיסונית 7-10, אשר כעת מאפשרת יישום הקליני של mRNAs שונה. באמצעות מיוצר באופן סינטטי mRNAs שונה, תמלילי גן רצויים יכולים להיות מועברים באופן זמני בvivo 11או במבחנה כדי לגרום לביטוי חלבון. MRNA הציג מתורגם בתנאים פיסיולוגיים על ידי מכונות תרגום הסלולריות. בשל חוסר האינטגרציה לתוך הגנום של התא הפונדקאי לעומת וקטורי ריפוי גנטי נגיפיים, הסיכון של oncogenesis נמנע 12,13. כך, טיפול באמצעות mRNA הסינתטי שונה יקבל קבלה קלינית טובה יותר בעתיד. כאן אנו מתארים פרוטוקול מפורט לייצור של ה- mRNA שונה, transfection של תאים עם mRNA וההערכה של ביטוי חלבון בתאי transfected (איור 1).

<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

המשלוח אקסוגני של קידוד RNA שליח סינטטי (mRNA) לזירוז סינתזה של חלבונים בתאים רצויים יש פוטנציאל עצום בתחומים של רפואת רגנרטיבית, ביולוגיה של תא בסיסי, טיפול במחלות, ותכנות מחדש של תאים. כאן, אנו מתארים צעד אחר צעד פרוטוקול לדור של mRNA שונה עם פוטנציאל מופחת חיסוני הפעלה ויציבות מוגברת, בקרת איכות של ה- mRNA מיוצר, transfection של תאים עם mRNA ואימות של ביטוי חלבון הנגרם על ידי זרימת cytometry. עד 3 ימים לאחר transfection יחיד עם eGFP mRNA, HEK293 תאי transfected לייצר eGFP. במאמר זה וידאו, הסינתזה של eGFP mRNA מתוארת כדוגמא. עם זאת, ההליך יכול להיות מיושם לייצור של ה- mRNA הרצוי האחר. שימוש בסינטטי שונה mRNA, יכולים להיגרם תאים לבטא את החלבונים הרצויים, שבו הם בדרך כלל לא מביעים זמניים.

באמצעות פלסמיד pcDNA 3.3 המכיל את ה- CDS של eGFP, הסינתזה של eGFP mRNA שונה הוקמה (איור 1). לאחר הגברה להוסיף על ידי PCR ופולי T-עוקב, להקה ברורה באורך של כ -1,100 נ&quot;ב הוא זוהה (איור 2). הגדלת זמן IVT Augmented התשואה של mRNA (איור 3). לאחר IVT, להקת mRNA ברורה באורך של כ -1,100 נ&q...

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

במאמר זה וידאו, אנו מתארים את הסינתזה במבחנה של mRNA שונה לזירוז של ביטוי חלבון בתאים.

במבחנת הסינתזה של mRNA השתנה לאינדוקציה של ביטוי חלבון בתאים אנושיים

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

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

24.9K Views.  University Hospital Tuebingen.  במאמר זה וידאו, אנו מתארים את הסינתזה במבחנה של mRNA שונה לזירוז של ביטוי חלבון בתאים. 

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

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

שימוש בחוץ גופית וצורה בתא לחקור מולקולה קטנה המושרה שינויים מבניים Pre-mRNA

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

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.

מבוסס-RNA תכנות מחדש של האנושי Fibroblasts הראשי לתוך תאי גזע Pluripotent המושרה

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

<meta charset="utf8"></head><body>פרוטוקולי CFPS עבור תאים סינתטיים מיקרו-ממודרים

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.

<meta charset="utf8"></head><body>מחבר זרקור: אופטימיזציה של מערכות CFPS לבניית תאים סינתטיים

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.

מערכת סינתזת חלבונים ללא תאים לבניית תאים סינתטיים

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 ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

הכנת חלבון הפקת תאים סינתטיים באמצעות שולפת תא חינם בקטריאלי, ליפוזומים והעברת אמולסיה

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

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.

פלטפורמת ביטוי נוח כללי לייצור חלבונים המופרשים מ תאים אנושיים

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.

העברה יעילה במיוחד של מקרופאגים ראשוניים עם מחוץ לגופית

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

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon1, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells2,3. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here.
Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells4, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs5-11. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.12 modified the shRNA expression vector pSUPER5 by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest.
Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome13,14 and apoptosis signaling15,16. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

A rapid and simple way to generate human cell lines with inducible and reversible cDNA overexpression or shRNA-mediated knock-down of the gene of interest. This method enables researchers to reliably and highly reproducibly manipulate cell lines that are difficult to alter by transient transfection methods or conventional knockdown/knockout strategies.

דור של שורות תאים אנושיות יציבות עם טטרציקלין-מושרה (ט ב) shRNA או ביטוי cDNA

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

במבחנת הסינתזה של mRNA השתנה לאינדוקציה של ביטוי חלבון בתאים אנושיים

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שימוש בחוץ גופית וצורה בתא לחקור מולקולה קטנה המושרה שינויים מבניים Pre-mRNA

מבוסס-RNA תכנות מחדש של האנושי Fibroblasts הראשי לתוך תאי גזע Pluripotent המושרה

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

מערכת סינתזת חלבונים ללא תאים לבניית תאים סינתטיים

מערכת סינתזת חלבונים ללא תאים לבניית תאים סינתטיים

מערכת סינתזת חלבונים ללא תאים לבניית תאים סינתטיים

הכנת חלבון הפקת תאים סינתטיים באמצעות שולפת תא חינם בקטריאלי, ליפוזומים והעברת אמולסיה

פלטפורמת ביטוי נוח כללי לייצור חלבונים המופרשים מ תאים אנושיים

העברה יעילה במיוחד של מקרופאגים ראשוניים עם מחוץ לגופית

דור של שורות תאים אנושיות יציבות עם טטרציקלין-מושרה (ט ב) shRNA או ביטוי cDNA