SV ist dankbar für die finanzielle Unterstützung von der NIH K22AI077353 und der Landenberger-Stiftung. KR dankt Mittel aus dem NIH (5SC1GM083754).

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Die Autoren haben nichts zu offenbaren.

Die in vitro-pre-mRNA 3 &#39;Spaltungsreaktion, in HeLa-Zellkernextrakten oder mit Spaltung Faktoren aus diesen Extrakten fraktioniert durchgeführt wird, hat die Identifizierung der Kernspaltungsfaktoren und deren Hauptkomplexe 16-21 aktiviert. Viele weitere Proteine ​​mit diesen Faktoren zugeordnet wurden kürzlich identifiziert 22 und der in-vitro-Reaktion kann weiterhin Licht auf, wie diese Proteine ​​tragen zur Reaktion zu vergießen. Vielleicht weil die Reaktion in vivo scheint cotranscriptional 23 sein, oder, da einige Faktoren können während der Extraktion und Dialyse verloren gehen, ist der Wirkungsgrad oft schlecht, und nur selten mehr als 30% Spaltung erreicht. Zusätzlich kann die Reaktionseffizienz plötzlich fallen von Tag zu Tag ohne ersichtlichen Grund. Daher ist es wichtig zu wissen, welche Schritte sind die kritischsten, insbesondere wenn versucht wird, um die Reaktion zu modifizieren oder viral infizierten HeLa-cel anzupassenls, zu anderen Zelltypen oder neue Substrate. Ohne Zweifel, die Qualität des Extraktes und die Frische der in vitro transkribierten RNA im Vordergrund stehen, ebenso wie die Notwendigkeit, gewissenhaft unter RNase-freien Bedingungen zu arbeiten. Die Gesundheit der Zellen an der Zeit, die sie für die Extraktion geerntet ist ebenfalls wichtig. Die Zellen sollten gesund erscheinen, sollten sie in weniger als 24 Stunden verdoppelt werden, sollten sie in oder nahe Mitte der log-Phase sein, und es sollten nur wenige tote Zellen oder andere unerklärliche Schmutz sein. Natürlich sollten die Zellen nicht durch Mykoplasmen oder andere Bakterien kontaminiert sein. Das Substrat sollte nicht mit zu hoher spezifischer Aktivität hergestellt werden, da dies zu offensichtlichen Abbau führen. Wenn eine ausreichend große Sequenz auf beiden Seiten der Spaltstelle betrachtet wird, und alternative Polyadenylierungsstelle ist berücksichtigt, die Anzahl der verschiedenen Poly (A)-Sequenz Signale im menschlichen Genom die Anzahl der Gene 24 zweifellos überschreitet </sbis&gt;. Da Schwankungen in der &quot;Stärke&quot; der verschiedenen Signale, die getestet wurden, bei der Anpassung des Verfahrens an eine neue Zelltyp, ist es am besten mit einer der stärkeren Polyadenylierungssignale, wie die häufig verwendeten Simian Virus 40 (begin SV40) spät Poly (A)-Signal 25. Die Kombination aus einem schwachen Poly (A)-Signalsequenz mit anderen als den Standard-HeLa-Zellen, Zellen können sich als frustrierend, und es ist besser, eine starke erste Substrat mit neuen Zellen oder unmodifizierten HeLa-Zellen mit einer potenziell schwachen Poly-A-Substrat zu verwenden. Auch nach so vielen Jahren der erfolgreichen Einsatz, gibt es noch kleinere Geheimnisse mit der In-vitro-3 &#39;Spaltungsreaktion verbunden. Zum Beispiel, warum wird Kreatinphosphat benötigt? Es hat sich gezeigt, dass der ursprüngliche Grund für die Aufnahme es - um die ATP-Pool zu regenerieren - ist nicht gültig 26. Interessanterweise kann es mit nur teilweiser Verlust der Aktivität, wenn die Kernextrakte sind weggelassenverwendet, jedoch wird zunehmend erforderlich, da der Extrakt wird in teilweise gereinigten Spaltungsfaktoren, die verwendet werden, um die Reaktion zu rekonstituieren fraktioniert. In der Tat, Phosphocholin, ein weiteres kleines Molekül mit einer Phosphatgruppe und einer positiv geladenen Amin, aber wahrscheinlich ohne in vivo Rolle in der Reaktion festgestellt worden ist wirksamer als Kreatinphosphat, zumindest in der rekonstituierten Reaktions 27 sein. PVA ist eine weitere Zutat, deren Bestimmung nicht umfassend erläutert. Es wird angenommen, dass ein Verdrängungsmittel sein, was zu einer effektiven Erhöhung der Spaltung Faktor Konzentration, aber auch andere drängen Mittel, wie Polyethylenglykol, nicht annähernd so gut funktionieren. Das Verständnis dieser Faktoren könnten zu einer verbesserten Effizienz führen, so dass die Erweiterung des Verfahrens auf weniger effiziente Zelltypen und RNA-Substrate und könnte Hinweise darauf, wie die verschiedenen Faktoren Arbeit ergeben.

Die Biosynthese der meisten reifen eukaryotischen Nachricht RNAs (mRNAs) erfordert mehrere posttranskriptionale Modifikationen wie Capping, Spleißen und Polyadenylierung. Diese Modifikationen sind in der Regel gekoppelt ist, um eine korrekte Verarbeitung 1 zu gewährleisten und die Stabilität der mRNA stark erhöhen. Das 3&#39;-Ende die Bildung von Säuger-prä-mRNAs durch endonukleolytische Spaltung des entstehenden RNA, gefolgt von Zugabe der Adenylat-Reste an den 5 &#39;erzeugt gespalten Produkt von Poly (A)-Polymerase (PAP) 2-4. Bei Säugetieren ist Spaltung durch ein Mehrkomponenten-Protein-Komplex durchgeführt, von etwa 800 kDa, die auf spezifische RNA-Sequenzen vor-montiert. Die Poly (A)-Signalsequenz, eine hoch konservierte kanonische Hexanukleotidsequenz AAUAAA, leitet die Spaltstelle bei etwa 10-30 nt stromabwärts. Diese Seite ist speziell von der Spaltung und Polyadenylierung Spezifität Faktor (CPSF) und dem 73 kD-Untereinheit der anerkanntenCSPF enthält die Endonukleaseaktivität. Die Spaltung Stimulationsfaktor (CstF) bindet eine degenerierte GU-oder U-reiche Element Sequenz stromabwärts des Poly (A)-Stelle. Erforderlich für die Spaltung der Spaltungssäugerfaktor I (cfim) und die Spaltung Säugerfaktor II (CFII). Cfim bindet die spezifischen UGUA (N)-Stellen in vor-Sequenzelemente (benutzt), die für eine Reihe von Genen, die definiert wurden, und scheint in wichtige physiologische Prozesse 5-8 einbezogen werden. In vitro RNA-Prozessierung Reaktionen sind allgemein durch die Verwendung von radioaktiv markierten RNA-Substrate 9-12 analysiert. Diese können durch Run-off-Transkription aus dem Bakteriophagen T7-oder SP6-Promotors synthetisiert werden. Bei der Untersuchung eine Polyadenylierungsstelle, die vorher nicht charakterisiert worden ist, ist es notwendig, um genomische DNA, anstatt cDNA zu verwenden, um das RNA-Substrat zu erzeugen, wie wichtig stromabwärts gelegenen Sequenzen in cDNA vorhanden sein könnten. Design Substrate mindestens 150 nt upstrea umfassenm und 50 nt stromabwärts von der Spaltstelle / Ende auf der reifen mRNA. Das Spaltprodukt wandert schneller als das Substrat; da jedoch andere Fragmente können durch nicht-spezifische Nuklease Aktion erzeugt werden, weist die Spezifität der Reaktion durch die Abhängigkeit von der korrekten Bearbeitung Signalfolgen überprüft werden. Daher RNA-Substrate mit einer Punktmutation in der Sequenz AAUAAA (zB AAGAAA) dienen als negative Kontrolle für die Spaltungsreaktion. Da eine geringe Menge radioaktiv markierten RNA wird für die Spaltungsreaktionen eingesetzt wird, kann in hoher Zahl in den meisten Kernextrakten RNasen problematisch sein, und die Wahl des Ausgangsmaterials für die Extraktzubereitung beschränken. HeLa-Zellen dazu neigen, niedrige endogene RNase enthalten, und somit in diesen Assays positiv. Die endonukleolytische Spaltung von RNA-Substrate in vivo und in vitro sofort von der Poly (A)-Additions, Do gefolgts die gespaltene Zwischen ist nicht in nachweisbaren Mengen vorhanden. Deshalb, um entweder eine bestimmte RNA-Sequenz oder Proteinen in einer Spaltungsreaktion beteiligt studieren, Experimente unter Bedingungen, die die Polyadenylierung verhindern geführt. Es gibt keine Abhängigkeit der Spaltung auf die Polyadenylierung, oder umgekehrt, so kann man Polyadenylierung, ohne die Spaltungsreaktion zu stoppen. Somit wird ATP mit einem Kettenabbruch Analogon, das die 3&#39;-Hydroxylgruppe fehlt, so daß nur ein einzelnes Nukleotid an der Poly (A)-Stelle eingebaut werden und nur gespalten RNA nachgewiesen werden kann ersetzt. In Anbetracht der Komplexität und hohe Besonderheit dieser Art von Assay, beschreiben wir eine detaillierte Videoprotokoll zu endonukleolytische Spaltung durch die Spaltungs / Poly (A) mRNA-Maschinen-Vorstufen in vitro zu studieren. Wir beschreiben, wie die zuständigen Kernextrakte herzustellen, erzeugen radioaktiv markierten RNA-Substrate, führen die Spaltungsreaktion und analysieren und interpretieren Sie das Ergebnisten Produkte. Fig. 1 zeigt ein Beispiel eines Substrats RNAs kodiert für das 3&#39;-Ende des HIV-1-prä-mRNAs für eine Spaltungsassay verwendet werden. Das 3&#39;-Ende des HIV-RNA-Genom ist von vielen wichtigen regulatorischen Sequenzen wie die Poly (A)-Stelle, einem G + U-reiche Region, und die Verwendung Element, die alle für eine effiziente Reifung der viralen mRNA-Transkripte 13 notwendig sind zusammen . In diesem Beispiel würden wir erwarten, dass die Eingangs RNA-Substrat auf 338 nt und 237 nt bei der Spaltung sein. Wenn Polyadenylierung durfte auftreten, würde ein Abstrich von Produkten zwischen 237 und 437 nt beobachtet werden.

<table border="0" cellpadding="0" cellspacing="0" width="757">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1L Celstir Flask</td>
			<td style="width:141px;">Wheaton</td>
			<td style="width:119px;">356884</td>
			<td style="width:281px;">Different sizes available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4 Position Slow Speed Stirrer</td>
			<td style="width:141px;">VWR</td>
			<td style="width:119px;">12621-076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Swinging Bucket Centrifuge</td>
			<td style="width:141px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td style="width:281px;">Allegra x-15R with SX4500 Rotor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultra Centrifuge</td>
			<td style="width:141px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td>Optima L-100 XP Ultra with SW41 Rotor</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Table Top Centrifuge 5417R</td>
			<td style="width:141px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td>Refridgerated</td>
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			<td height="21" style="height:21px;width:216px;">Thermomixer incubator</td>
			<td style="width:141px;">Eppendorf</td>
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			<td height="21" style="height:21px;width:216px;">250ml Conicle Tubes</td>
			<td style="width:141px;">Corning</td>
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			<td height="21" style="height:21px;width:216px;">50ml Conicle Tubes</td>
			<td style="width:141px;">BD Falcon</td>
			<td style="width:119px;">352098</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultra-clear Centrifuge Tubes</td>
			<td style="width:141px;">Beckman Coulter</td>
			<td style="width:119px;">344059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">JOKLIK Modified MEM</td>
			<td style="width:141px;">Lonza</td>
			<td style="width:119px;">04-719Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:141px;">Atlas&#160;</td>
			<td style="width:119px;">F-0500-A</td>
			<td>Heat inactivated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-Glut:Pen:Strep</td>
			<td style="width:141px;">Gemini Bio-Products</td>
			<td style="width:119px;">400-110</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1M Tris-HCl pH 8.0</td>
			<td style="width:141px;">Mediatech</td>
			<td style="width:119px;">46-031-CM</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Chloride</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP214-500</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium Chloride</td>
			<td style="width:141px;">MP Biomedicals</td>
			<td style="width:119px;">194844</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP310-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DTT</td>
			<td style="width:141px;">Alexis Biomedicals</td>
			<td style="width:119px;">280-001-G-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP229-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5M Sodium Chloride Solution</td>
			<td style="width:141px;">Mediatech</td>
			<td style="width:119px;">46-032-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA 0.5M Solution</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">E7889-100ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP120-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PMSF</td>
			<td style="width:141px;">Thermo Scientific</td>
			<td style="width:119px;">36978</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ammonium Sulfate</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">A702-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">cOmplete EDTA-Free Protease inhibitor cocktail tablets</td>
			<td style="width:141px;">Roche</td>
			<td style="width:119px;">04 693 132 001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Slide-A-Lyzer Dialysis Cassettes Kit</td>
			<td style="width:141px;">Thermo Scientific</td>
			<td style="width:119px;">66372</td>
			<td>MWCO 7000 0.5ml-3ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">15ml Dounce Tissue grinder set</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9938-1SET</td>
			<td>Different sizes available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Expand High Fidelity PCR Kit</td>
			<td style="width:141px;">Roche</td>
			<td style="width:119px;">11 732 650 001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10mM dNTP Mix</td>
			<td style="width:141px;">Invitrogen</td>
			<td style="width:119px;">Y02256</td>
			<td>10mM each nucleotide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MaxiScript SP6/T7 Kit</td>
			<td style="width:141px;">Ambion</td>
			<td style="width:119px;">AM1322</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">m7G(5&#39;)ppp(5&#39;) G RNA Cap</td>
			<td style="width:141px;">New England Biolabs</td>
			<td style="width:119px;">S1404S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Century Marker Template Plus</td>
			<td style="width:141px;">Ambion</td>
			<td style="width:119px;">AM7782</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Easytides UTP [alpha-32p]-250uCi</td>
			<td style="width:141px;">Perkin Elmer</td>
			<td style="width:119px;">BLU507H250UC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel loading buffer II</td>
			<td style="width:141px;">Ambion</td>
			<td style="width:119px;">8546G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DEPC treated water</td>
			<td style="width:141px;">Ambion</td>
			<td style="width:119px;">AM9906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10x TAE</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP13354</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10x TBE</td>
			<td style="width:141px;">Ameresco Life Sciences</td>
			<td style="width:119px;">0658-4L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10x PBS</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP399-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP169-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ammonium Persulfate</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;">161-0700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TEMED</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP150-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ammonium Acetate</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">A637-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">40% 19:1 Acrylamide:Bis-acrylamide</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;">161-0144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycogen</td>
			<td style="width:141px;">Roche</td>
			<td style="width:119px;">10 901 393 001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100% Absolute Ethanol 200 Proof</td>
			<td style="width:141px;">Acros</td>
			<td style="width:119px;">61509-0040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acid Phenol-Chloroform</td>
			<td style="width:141px;">Ambion</td>
			<td style="width:119px;">9720</td>
			<td>For RNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scintilation Fluid</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">SX18-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rnase Inhibitor</td>
			<td style="width:141px;">Promega</td>
			<td style="width:119px;">N261B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Poly (Vinyl alcohol) PVA</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8136-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Creatine Phosphate</td>
			<td style="width:141px;">Calbiochem</td>
			<td style="width:119px;">2380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100mM dATP</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP2560-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:141px;">Acros</td>
			<td style="width:119px;">23042-5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Proteinase K</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP1700-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adjustable Sequencing Unit</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z351881-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Binder Clips</td>
			<td style="width:141px;">Office Depot</td>
			<td style="width:119px;">838-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">20cmx42cm glass plates</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z352543</td>
			<td>1SET</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">20cmx22cm glass plates</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z35252-7</td>
			<td>1SET</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">20cmx42cm Aluminum Cooling Plates</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z352667</td>
			<td>1EA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.4mmx22cm Spacers</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z35230-6</td>
			<td>1SET</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.4mmx42cm Spacers</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z352314-1</td>
			<td>1SET</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">8-well Comb</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z35195-4</td>
			<td>1EA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">16-well Comb</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z351962</td>
			<td>1EA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">32-well Comb</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z351970</td>
			<td>1EA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel Repel Coating</td>
			<td style="width:141px;">C.B.S. Scientific</td>
			<td style="width:119px;">SGR-0401</td>
			<td>or SGR-0101 for individual bottle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel Loading Tips</td>
			<td style="width:141px;">Rainin</td>
			<td style="width:119px;">GT-10-4</td>
			<td>0.1-10uL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sequencing PowerPac HV</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;">PowerPac HV</td>
			<td>5000V/500mA/400W</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel Dryer Model 583</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;">Model 583</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hydrotech Vacuum Pump for gel dryer</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glogos II Glow-in-the-dark Markers</td>
			<td style="width:141px;">Agilent</td>
			<td style="width:119px;">420201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Film 8x10</td>
			<td style="width:141px;">Midsci</td>
			<td style="width:119px;">BX810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Film 14x17</td>
			<td style="width:141px;">Phenix</td>
			<td style="width:119px;">F-BX1417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Autoradiography Cassette</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">FBCA 1417</td>
			<td>8x10 size available</td>
		</tr>
	</tbody>
</table>

analysis of rna processing reactions using cell free systems: 3' end cleavage of pre-mrna substrates in vitro

Das 3&#39;-Ende von Säuger mRNAs nicht durch abrupte Abbruch der Transkription durch RNA-Polymerase II (RNPII) ausgebildet ist. Stattdessen synthetisiert RNPII Vorläufer-mRNA über das Ende des reifen RNAs, und ein aktiver Prozess, der Endonuklease-Aktivität wird an einer bestimmten Stelle erforderlich. Die Spaltung der Vorläufer-RNA tritt normalerweise 10-30 nt stromabwärts von der Konsens polyA-Stelle (AAUAAA), nachdem die CA-Dinukleotide. Proteine ​​aus der Spaltungskomplex eine multifaktorielle Protein-Komplex von etwa 800 kDa, erfüllen diese spezifischen Nuklease-Aktivität. Spezifische RNA-Sequenzen stromaufwärts und stromabwärts von der polyA-Stelle steuern die Einstellung der Spalt komplex. Unmittelbar nach der Spaltung werden prä-mRNAs durch die Poly-A-Polymerase (PAP) polyadenyliert zu produzieren stabile RNA-Nachrichten zu reifen. Verarbeitung von dem 3&#39;-Ende eines RNA-Transkripts können unter Verwendung von Zellkernextrakten mit spezifischen radioaktiv markierten RNA-Substrate untersucht. In Summe ein lang 32 &lt;/ Sup&gt; P-markierten ungespaltenen Vorläufer RNA wird mit Kernextrakten in vitro inkubiert und Spaltung wird durch Gelelektrophorese und Autoradiographie untersucht. Wenn der richtig Spaltung auftritt, eine kürzere 5 &#39;gespalten Produkt nachgewiesen und quantifiziert. Hier beschreiben wir die Spaltungstest im Detail, wobei als ein Beispiel, das den 3&#39;-End-Verarbeitung von HIV-1 mRNAs.

Repräsentative Ergebnisse einer Spaltungsassay der RNA Poly (A)-Stellen von HIV-1 (Fig. 2). Wir können die ungespaltene RNA-Substrat, das am langsamsten wandernde Bande an der Spitze des Gels zu beobachten. Die spezifische gespaltene Produkt ist die intensivste kürzere Fragment Band, die schneller in der Gel bei der erwarteten Größe läuft und ist speziell aus der Spaltung Assay der mutpoly (A) Kontrolle, die eine Punktmutation in der Poly-A-Sequenz (mutPolyA) RNA enthält abwesend Substrat. Abbauprodukte des Eingangs Substrat kann manchmal beobachtet werden. Quantifizierung der Spaltungsaktivität kann durch densitometrische Plot des Verhältnisses der ungespaltenen zu spaltende RNA normalisiert auf 100% des nicht gespaltenen RNA bestimmt werden. <img alt="Figur 1" fo:content-width="6in" src="/files/ftp_upload/51309/51309fig1highres.jpg" width="600" /> 1. Schematische Darstellung des HIV-vorgemRNA 3&#39;-Ende Verarbeitung Substrate und erwarteten Produkte von in-vitro-Spaltung und Polyadenylierung Reaktionen. Die Spaltstelle, die Dinukleotid-CA-liegt 25 nt stromabwärts von der polyA Website AAUAAA. LTR-Regionen: U3 (unique 3 &#39;Sequenz), R (wiederholte Sequenz) und U5 (unique 5&#39; Sequenz). <img alt="Figur 2" fo:content-width="5in" src="/files/ftp_upload/51309/51309fig2highres.jpg" width="500" /> 2. (A) Poly (A)-Stelle Spaltung. 32 P-markierten RNA, die die Poly (A) des HIV-1 und eine Punktmutation des Poly (A)-Signal, die Spaltung (mutPolyA) hebt wurden in Zellkernextrakten inkubiert von HeLa-S3-Zellen oder BSA als negative Kontrolle. Fettdruck Pfeil zeigt 5 &#39;gespalten Produkt. Das 3-Fragment läuft oft aus dem Gel oder abgebaut und nicht immer sichtbar. (B) Densitometrische Grundstück von der Spaltung Aktivitätenty ausgedrückt als Verhältnis der ungespaltenen gespalten RNA normalisiert auf 100% des nicht gespaltenen RNA. <table border="0" cellpadding="0" cellspacing="0" width="1032"><tbody><tr><td> Puffer A (100 ml) </td><td> 10 mM Tris (pH 7,9), 1,5 mM MgCl 2, 10 mM KCl, 0,5 mM Dithiothreitol (DTT). </td><td> In DTT kurz vor der Verwendung von Puffer A während der Extraktion. 1 &amp;mgr; l 1 M DTT pro 1 ml Puffer A </td></tr><tr><td> Puffer C (50 ml) </td><td> 20 mM Tris (pH 7,9), 25% (v / v) Glycerin, 0,42 M NaCl, 1,5 mM MgCl 2, 0,2 mM Ethylendiamintetraessigsäure (EDTA), 0,5 mM DTT, 0,5 mM Phenylmethylsulfonylfluorid (PMSF). </td><td> Am Morgen der Extraktionen Hinzufügen eines Protease-Inhibitor-Cocktail Tablette. </td></tr><tr><td> Buffer D 50 (4.2 L) </td><td> 20 mM Tris (pH 7,9), 20% (v / v) Glycerin, 0,2 mM EDTA, 0,5 mM DTT, 50 mM Ammoniumsulfat. </td><td> HEPES-KOH kann in Tris-Puffer A, C und D substituiert sein kann </td></tr><tr><td&gt; RNA Elution Buffer </td><td> 0,5 M Amonium Acetat, 10 mM MgCl 2, 1 mM EDTA, 0,2% Natriumdodecylsulfat (SDS) </td><td> Lagern bei Raumtemperatur. </td></tr><tr><td> Anschlagpuffer (ETS) </td><td> 10 mM Tris (pH 8,0), 10 mM EDTA, 0,5% SDS </td><td> Lagern bei Raumtemperatur. </td></tr><tr><td> Die Spaltung Ladepuffer </td><td> 90% Formamid, 5 mM EDTA, pH 8, 0,025% Bromphenolblau </td><td> Lagerung bei -20 ° C. </td></tr></tbody></table> Tabelle 1. Zusammensetzung der Puffer.

Watch this Scientific Journal Video about Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro at JoVE.com

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

RNA-Polymerase II synthetisiert eine Vorläufer-RNA, die über das 3&#39;-Ende der reifen mRNA erstreckt. Das Ende der reifen RNA cotranscriptionally über die Endonuklease-Aktivität der Spaltungskomplex erzeugt wird, an einer Stelle, die durch RNA-Sequenzen bestimmt. Hier haben wir detailliert die Methode, um Spaltungsreaktionen in vitro zu studieren.

Die Analyse der RNA-Prozessierung Reaktionen mit Handy Free Systems: 3 &#39;Ende Spaltung von prä-mRNA-Substrate<em&gt; In-vitro-</em

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

analysis-rna-processing-reactions-using-cell-free-systems-3-end

Nanyang Technological University

Research

JoVE Journal

Biology

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

12.7K Aufrufe.  The Scripps Research Institute.  RNA-Polymerase II synthetisiert eine Vorläufer-RNA, die über das 3'-Ende der reifen mRNA erstreckt. Das Ende der reifen RNA cotranscriptionally über die Endonuklease-Aktivität der Spaltungskomplex erzeugt wird, an einer Stelle, die durch RNA-Sequenzen bestimmt. Hier haben wir detailliert die Methode, um Spaltungsreaktionen in vitro zu studieren. 

Serie: Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Video bioinformatischen Analyse von humanen embryonalen Stammzellen Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Forschung

Biologie

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

Visualisierung der MG53-vermittelte Cell Membrane Repair mit In vivo Und In-vitro- Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina&#x2019;s genome analyzer performs sequencing of a large number (&gt; 107) of relatively short sequence reads (&lt; 150 bp).The &quot;paired end&quot; approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo transcriptome assembly.
One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described1, 2 but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published3, 4 but require microgram amounts of starting total RNA.
Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing5, where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined &quot;T7LA&quot; (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome6 and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples7.

Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

Single Read und Paired End mRNA-Seq Illumina Bibliotheken von 10 Nanogramm Gesamt-RNA

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

In vitro Transkription und Capping von Gaussia-Luciferase mRNA von HeLa-Zelle Transfektion Gefolgt

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

Zellspezifische Analyse<em&gt; Arabidopsis</em&gt; Blätter mit Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

Substrat Generation für Endonukleasen der CRISPR / Cas-Systeme

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e. total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

Metabolische Markierung der neu transkribierten RNA für High Resolution Gene Expression Profiling von RNA Synthese, Verarbeitung und Decay in Zellkultur

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene ...

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed &#8220;in vitro human cell free expression systems&#8221;. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 &#181;l of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer&#8217;s Cleft &#8211; 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 &#181;l of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford&#8217;s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

Basierend HeLa Zellfreie Expressionssysteme zur Expression von<em&gt; Plasmodium</em&gt; Rhoptry Proteine

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

Ein zellfreien Assay zu Chromatindekondensation Studieren am Ende der Mitose

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

Nachweis von RNA-bindenden Proteinen durch<em&gt; In Vitro</em&gt; RNA Pull-down in Adipocyte Kultur

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...