Diese Arbeit wurde von Anpassungsfähig und Seamless Technologie-Transfer-Programm durch zielorientierte F &amp; E, Japan Science and Technology Agency, die Wissenschaft Forschungsförderungsfonds von der Förderung und den gegenseitigen Aid Corporation für Privatschulen von Japan unterstützt, und die JSPS KAKENHI Grants-Nummern 24300342 und 15H04313 .

1. Aufbau eines Heptamer-Typ sgRNA Bibliothek <ol><li> Wählen einer Teilmenge der 16.384 (4 7) 7-nt - Sequenzen , wenn der umfassende Bibliothek konstruiert werden soll. HINWEIS: Die Sequenzen können statistische und / oder gestaltet sein, spezifische zelluläre RNAs abzuzielen. Für letztere hairpin - Strukturen ähnlich der T-Arm von tRNA in einer Ziel - RNA mit Hilfe eines geeigneten Computerprogramms zu finden und / oder optisch und wählen Sequenzen komplementär zu 7-nt - Sequenzen unmittelbar stromabwärts der Haarnadelstrukturen 4,10, 17,18. </li><li> Synthetisieren jedes der ausgewählten Heptamere als ein vollständig 2&#39;- O -methylated, 5&#39;- und 3&#39;-phosphorylierter RNA mit einem DNA / RNA - Synthesizer. Verwenden Sie die Phosphoramidit-Methode auf dem Glas mit kontrollierten Poren (CPG) Unterstützung, und lassen Sie das 5&#39;-terminale 4,4&#39;-Dimethoxytrityl (DMT) Gruppe im letzten Zyklus zu schützen (die Zugabe des Phosphat &#39;5) der Synthese 23. HINWEIS: Benutzerdefinierte synth e tisches RNA kann auch von Oligonukleotid-Lieferanten erhalten werden. </li><li> Nach Abschluss der letzten Synthesezyklus, übertragen Sie die CPG mit dem synthetisierten Oligonukleotid aus der Säule in eine Glasampulle mit einem Schraubverschluss Teflon-Liner, 1 ml von 29% Ammoniumhydroxid und brüten sie bei 55 ° C für 8 h auf das Oligonucleotid von dem CPG abzuspalten und die Schutzgruppen von den Basen und Phosphaten entfernen. </li><li> Verdunsten Ammoniumhydroxid mit einem Zentrifugal-Verdampfer bei 40 ° C und wieder aufzulösen das Oligonucleotid in 0,2 ml 0,1 M n-hexylammonium acetat. </li><li> Reinige den synthetischen Oligonukleotids durch Umkehrphasen-Hochleistungs-Flüssigkeits-Chromatographie unter Verwendung eines Polystyrol-Divinylbenzol-Säule mit einem Puffer, der 10-50% Acetonitril / 0,1 M n-hexylammonium acetat. Führen Sie die Säule bei 2,0 ml / min für 25 min bei 80 ° C. </li><li> Hinzufügen 0,25-0,5 ml konzentrierter Essigsäure zu der fraktionierten Probe (1-2 ml), eine 20% ige Essigsäurelösung zu machen, und Incubate diese Lösung für 1 h bei Raumtemperatur die DMT-Gruppe zu entfernen. </li><li> Trocknen der Probe mit einem Zentrifugal-Verdampfer bei 40 ° C, wieder aufzulösen sie in 1 ml von 29% igem Ammoniumhydroxid, und Inkubieren der Lösung für 15 min bei Raumtemperatur die Seitenkette des 5&#39;-Phosphat zu entfernen. </li><li> Reduzieren das Volumen (1 ml) der Probe auf etwa 0,3 ml mit einem Zentrifugal-Verdampfer bei 40 ° C. </li><li> Dialysieren die gesamte konzentrierte Probe gegen destilliertes Wasser (500 ml) über Nacht einen Dialyseschlauch mit einem Molekulargewichts-Cut-off 1000 bei 4 ° C verwendet wird. </li><li> Übertragen die dialysierte Probe aus dem Dialyseschlauch in ein 1,5-ml-Röhrchen mit einer Pipette, trocknen mit einem Zentrifugal-Verdampfer bei 40 ° C und anschließend wieder aufzulösen es in Wasser für Injektionszwecke geeignetem Wasser, dessen Volumen sollte klein genug, um eine von mehr als 100 uM Lösung herzustellen. </li><li> Messen, um eine optische Dichte der RNA bei 260 nm mit einem Spektrophotometer und stellen seine Konzentration auf 100uM von Wasser zur Injektion-grade Zugabe von Wasser. Speichern die RNA-Lösung bei unter -20 ° C vor dem Gebrauch. </li></ol> 2. Screening der sgRNA Bibliothek für Zell Viability <ol><li> Vorbereitung 10 3 Zellen / 99 ul / Vertiefung (beispielsweise HL60 und RPMI-8226) auf einer Schale mit 96 Vertiefungen in RPMI-1640 - Medium mit 10% fötalem Rinderserum und 1% Penicillin-Streptomycin - Lösung. Bereiten Vertiefungen, die die Medien nur für Untergrundsubtraktion. Zur Vermeidung von Randeffekten, fügen Sie steriles Wasser zu leeren Brunnen rund um die Probenvertiefungen. </li><li> 1 ul Heptamers-Typ sgRNA (100 &amp; mgr; M), gelöst in Wasser für Injektionszwecke geeignetem Wasser zu jeder Vertiefung. Assay jedes sgRNA in dreifacher Ausfertigung. Inkubieren der Platte bei 37 ° C in einem 5% CO 2 befeuchteten Inkubator für 3 Tage. </li><li> Eine geeignete Menge eines handelsüblichen WST-8-Lösung in jede Vertiefung. Inkubieren der Platte bei 37 ° C im selben Inkubator für 1 bis 4 Stunden. </li><li> Messen Sie die Absorption einT 450 nm mit einem Mikroplattenleser. HINWEIS: In den meisten Fällen Linearität zwischen Extinktion und Lebendzellzahl wird beibehalten werden, es sei denn der Wert Extinktion 1,0 übersteigt. </li></ol> 3. Bewertung der effektiven sgRNAs durch Durchflusszytometrie <ol><li> Vorbereitung 10 3 Zellen / 99 ul / Vertiefung (beispielsweise HL60) auf einer Schale mit 96 Vertiefungen in RPMI-1640 - Medium mit 10% fötalem Rinderserum und 1% Penicillin-Streptomycin - Lösung. Vorbereitung mindestens 10 Vertiefungen für eine sgRNA getestet werden. Zur Vermeidung von Randeffekten, fügen Sie steriles Wasser zu leeren Brunnen rund um die Probenvertiefungen. </li><li> 1 ul Heptamers-Typ sgRNA (100 &amp; mgr; M), gelöst in Wasser für Injektionszwecke geeignetem Wasser zu jeder Vertiefung. Inkubieren der Platte bei 37 ° C in einem 5% CO 2 befeuchteten Inkubator für 3 Tage. </li><li> Sammeln Sie die mit dem gleichen sgRNA behandelten Zellen von mindestens 10 Brunnen in einen 5-ml-Röhrchen mit einer Pipette. Drehen Sie das Röhrchen 5 min in einer Zentrifuge bei 300 · g, und entsorgen Sie die Medien. </li><li> 2 ml 1x phosphatgepufferter Kochsalzlösung (PBS) mit dem Rohr und resuspendieren der Zellen. Drehen Sie das Röhrchen 5 min in einer Zentrifuge bei 300 g und verwerfen PBS. Zugabe von 150 ul 1x Annexin V-Bindungspuffer auf das Rohr und die Zellen erneut zu suspendieren. </li><li> In 2 ul Phycoerythrin (PE) konjugiertem Annexin V-Lösung und 1 ul 7-Aminoactinomycin D (7AAD) Lösung in das Röhrchen, mischen sie gut, und Inkubation die Röhrchen für 15 Minuten bei Raumtemperatur im Dunkeln. Analysieren Sie die Probe ein Durchflusszytometer 24 verwendet wird . </li></ol>

<ol>
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Chem. 280, 22326-22334 (2005).</li><li>Tamura, M., Nashimoto, C., Miyake, N., Daikuhara, Y., Ochi, K., Nashimoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+mRNA+cleavage+by+3&#8242;+tRNase+under+the+direction+of+2&#8242;-O-methyl+RNA+heptamers.">Intracellular mRNA cleavage by 3&#8242; tRNase under the direction of 2&#8242;-O-methyl RNA heptamers.</a> Nucleic Acids Res. 31, 4354-4360 (2003).</li><li>Habu, Y., 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=Inhibition+of+HIV-1+gene+expression+by+retroviral+vector-mediated+small-guide+RNAs+that+direct+specific+RNA+cleavage+by+tRNase+ZL.">Inhibition of HIV-1 gene expression by retroviral vector-mediated small-guide RNAs that direct specific RNA cleavage by tRNase ZL.</a> Nucleic Acids Res. 33, 235-243 (2005).</li><li>Nakashima, A., 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=Gene-silencing+by+the+tRNA+maturase+tRNase+ZL+under+the+direction+of+small+guide+RNA.">Gene-silencing by the tRNA maturase tRNase ZL under the direction of small guide RNA.</a> Gene Ther. 14, 78-85 (2007).</li><li>Elbarbary, R. A., Takaku, H., Tamura, M., Nashimoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+vascular+endothelial+growth+factor+expression+by+TRUE+gene+silencing.">Inhibition of vascular endothelial growth factor expression by TRUE gene silencing.</a> Biochem. Biophys. Res. Commun. 379, 924-927 (2009).</li><li>Sano, T., Takahashi, M., Nozaki, T., Takahashi, Y., Tamura, M., Nashimoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+utility+of+heptamer-type+sgRNA+for+TRUE+gene+silencing.">Expanding the utility of heptamer-type sgRNA for TRUE gene silencing.</a> Biochem. Biophys. Res. Commun. 416, 427-432 (2011).</li><li>Iizuka, S., Oridate, N., Nashimoto, M., Fukuda, S., Tamura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+inhibition+of+head+and+neck+squamous+cell+carcinoma+cells+by+sgRNA+targeting+the+cyclin+D1+mRNA+based+on+TRUE+gene+silencing.">Growth inhibition of head and neck squamous cell carcinoma cells by sgRNA targeting the cyclin D1 mRNA based on TRUE gene silencing.</a> PLoS One. 9 (114121), (2014).</li><li>Takahashi, 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=Elimination+of+specific+miRNAs+by+naked+14-nt+sgRNAs.">Elimination of specific miRNAs by naked 14-nt sgRNAs.</a> PLoS One. 7 (38496), (2012).</li><li>Takahashi, 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=A+naked+RNA+heptamer+targeting+the+human+Bcl-2+mRNA+induces+apoptosis+of+HL60+leukemia+cells.">A naked RNA heptamer targeting the human Bcl-2 mRNA induces apoptosis of HL60 leukemia cells.</a> Cancer Lett. 328, 362-368 (2013).</li><li>Watanabe, 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=Induction+of+apoptosis+of+leukemic+cells+by+TRUE+gene+silencing+using+small+guide+RNAs+targeting+the+WT1+mRNA.">Induction of apoptosis of leukemic cells by TRUE gene silencing using small guide RNAs targeting the WT1 mRNA.</a> Leukemia Res. 37, 580-585 (2013).</li><li>Elbarbary, R. A., 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=Modulation+of+gene+expression+by+human+cytosolic+tRNase+ZL+through+5&#8242;-half-tRNA.">Modulation of gene expression by human cytosolic tRNase ZL through 5&#8242;-half-tRNA.</a> PLoS One. 4, 5908 (2009).</li><li>Elbarbary, R. A., 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=Human+cytosolic+tRNase+ZL+can+downregulate+gene+expression+through+miRNA.">Human cytosolic tRNase ZL can downregulate gene expression through miRNA.</a> FEBS Lett. 583, 3241-3246 (2009).</li><li>Ninomiya, 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=Potential+small+guide+RNAs+for+tRNase+ZL+from+human+plasma,+peripheral+blood+mononuclear+cells,+and+cultured+cell+lines.">Potential small guide RNAs for tRNase ZL from human plasma, peripheral blood mononuclear cells, and cultured cell lines.</a> PLoS One. 10, 0118631 (2015).</li><li>Takahashi, 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=Screening+of+a+heptamer-type+sgRNA+library+for+potential+therapeutic+agents+against+hematological+malignancies.">Screening of a heptamer-type sgRNA library for potential therapeutic agents against hematological malignancies.</a> Leukemia Res. 38, 808-815 (2014).</li><li>Shivarov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRUE+gene+silencing+for+hematologic+malignancies.">TRUE gene silencing for hematologic malignancies.</a> Leukemia Res. 38, 729 (2014).</li></ol>

Niigata University of Pharmacy and Applied Life Sciences has a pending patent application on a subset of the heptamer-type sgRNAs.

In most cases, fully 2&#8242;-O-methylated, 5&#8242;- and 3&#8242;-phosphorylated heptamer-type sgRNAs dissolved in water-for-injection-grade water (in the concentration of 100 &#181;M) appear to be stable at below -20 &#176;C for at least one year, judging from their activity to induce apoptosis in cancer cells. However, their stability would change depending on their sequence, quality, and purity. In some cases, 5&#8242;- or 3&#8242;-phosphate of a part of sgRNA molecules appears to be removed spontaneously du...

TRUE Gen - Silencing (genannt nach tR Nase Z L - u e fficacious gene silencing tilizing) ist einer der RNA-directed gene silencing Technologien 1. Diese Technologie wurde auf die Eigenschaften von tRNase Z L (oder 3 &#39;tRNase), tRNA 3&#39; - Prozessierung Endoribonuclease entwickelt basiert: es jedes Ziel - RNA an einer erwarteten Stelle unter der Leitung eines künstlichen kleinen Führungs RNA spalten kann (sgRNA) durch eine Anerkennung pre-tRNA-ähnlichen oder Mikro pre-tRNA-ähnlichen Struktur zwischen der Ziel - RNA gebildet , und der sgRNA 2-9. sgRNA, die in der Regel 7-31 nt in der Länge, ist in vier Gruppen eingeteilt, 5&#39;-Hälfte-tRNA, RNA Heptamer, 14-nt RNA linear und hook RNA. Wir haben die Wirksamkeit von TRUE gene silencing demonstriert durch verschiedene künstlich gestaltete sgRNAs Einführung in die Zellen 10 leben - 15. Wir haben auch gezeigt, dass sgRNAs durch Zellen wi aufgenommen werden kann,thout jegliche Transfektionsreagenzien und können ihre Ziel - RNA - Niveaus downregulate und / oder Apoptose in menschlichen Krebszellen induzieren 16 - 18. TRUE Gen - Silencing erscheint auf dem intra- und interzellulären Genregulationsnetz System über tRNase Z L und natürliche sgRNAs 19 zu arbeiten - 21. Wir haben eine sgRNA Bibliothek mit 156 Heptamers-Typ sgRNAs konstruiert 22 für potentielle therapeutische Heptamers-Typ sgRNAs für Blutkrebs zu suchen. Wir haben es für die Wirkung auf die Lebensfähigkeit von menschlichen Myelom und Leukämie-Zellen gescreent, und fand, daß 20 von ihnen effizient Apoptose in zumindest einer der Krebszelllinien induzieren kann. Und 4 von ihnen haben gezeigt, dass Tumorwachstumsraten in Maus-Xenograft Experimente reduzieren. Hier präsentieren wir ein Protokoll für die Vorführung eines Heptamers-Typ sgRNA Bibliothek für potenzielle therapeutische Medikamente gegen Blutkrebs. Das Protokoll enthält, wiezu konstruieren, die sgRNA Bibliothek, wie die Auswirkungen der einzelnen sgRNA auf die Lebensfähigkeit der Zellen zu bewerten und wie Zytometrie die effektiven sgRNAs durch die Strömung weiter auszuwerten. Analyse für sgRNA der zellulären Ziel - RNAs und Bewertung der effektiven sgRNAs in Modellen Maus - Xenotransplantat kann nach herkömmlichen Verfahren durchgeführt werden 17,22, obwohl diese in diesem Protokoll nicht enthalten.

<table border="0" cellpadding="0" cellspacing="0" width="948">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Custom heptamer RNA</td>
			<td style="width:167px;">Nippon Bioservice</td>
			<td style="width:144px;"></td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OligoSep Prep HC Cartridge</td>
			<td style="width:167px;">Transgenomic</td>
			<td style="width:144px;">99-3860</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Water-for-injection-grade Water</td>
			<td style="width:167px;">Otsuka Pharmaceutical&#160;</td>
			<td style="width:144px;">7131400A2129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI-1640 medium</td>
			<td style="width:167px;">Wako</td>
			<td style="width:144px;">189-02025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td style="width:167px;">SIGMA-ALDRICH</td>
			<td style="width:144px;">172012-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Penicillin-Streptomycin Mixed Solution</td>
			<td style="width:167px;">Nacalai Tesque</td>
			<td style="width:144px;">26253-84</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 Well Plate</td>
			<td style="width:167px;">Greiner Bio-one</td>
			<td style="width:144px;">655 180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Counting Kit-8</td>
			<td style="width:167px;">DOJINDO</td>
			<td style="width:144px;">343-07623</td>
			<td>WST-8 solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate Reader, Sunrise Thermo RC-R</td>
			<td style="width:167px;">TEKAN</td>
			<td style="width:144px;">510-82851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS Round-Bottom Tube</td>
			<td style="width:167px;">BD Falcon</td>
			<td style="width:144px;">60819-820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline</td>
			<td style="width:167px;">SIGMA-ALDRICH</td>
			<td style="width:144px;">P7059-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE Annexin V Binding Buffer</td>
			<td style="width:167px;">BD Biosciences</td>
			<td style="width:144px;">51-66121E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE Annexin V</td>
			<td style="width:167px;">BD Biosciences</td>
			<td style="width:144px;">51-65875X</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7AAD</td>
			<td style="width:167px;">SIGMA-ALDRICH</td>
			<td style="width:144px;">A9400-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow Cytometer, FACSCalibur</td>
			<td style="width:167px;">BD Biosciences</td>
			<td style="width:144px;">342973</td>
			<td></td>
		</tr>
	</tbody>
</table>

true gene silencing: screening of a heptamer-type small guide rna library for potential cancer therapeutic agents

TRUE Gen - Silencing (genannt nach tR Nase Z L - u e fficacious gene silencing tilizing) ist eine von der RNA-gerichtete Gen - Silencing - Technologien, die eine künstliche kleine Führungs RNA (sgRNA) verwendet tRNA 3&#39; - Prozessierung Endoribonuklease zu führen, tRNase Z L eine Ziel-RNA zu erkennen. sgRNAs kann durch Zellen ohne Transfektionsreagentien aufgenommen werden und können ihre Ziel-RNA-Niveaus und / oder induzieren Apoptose in menschlichen Krebszellen downregulate. Wir haben eine sgRNA Bibliothek mit 156 Heptamer-Typ sgRNAs für die Wirkung auf die Lebensfähigkeit von menschlichen Myelom und Leukämie-Zellen gescreent, und fand, daß 20 von ihnen effizient Apoptose in zumindest einer der Krebszelllinien induzieren kann. Hier präsentieren wir ein Protokoll für die Vorführung eines Heptamers-Typ sgRNA Bibliothek für potenzielle therapeutische Medikamente gegen Blutkrebs. Das Protokoll enthält, wie die sgRNA Bibliothek zu konstruieren, wie die Auswirkungen der einzelnen sgRNA auf die Lebensfähigkeit der Zellen zu bewerten und wie fueitere bewerten die effektiven sgRNAs mittels Durchflusszytometrie. Rund 2.000 Treffer wäre zu erwarten, durch das Screening der Full-Scale sgRNA Bibliothek von 16.384 Heptamere zusammengesetzt zu erhalten.

Die sechs Heptamer-Typ sgRNAs 5&#39;-AUCUUCA-3 &#39;(H1885), 5&#39;-ACACACA-3&#39; (H3277), 5&#39;-GGGGGCG-3 &#39;(H10927), 5&#39;-GGGGCCC-3&#39; (H10944), 5&#39;-GCCCCCG-3 &#39;(H12287) und 5&#39;-CACCAGC-3&#39; (H13260) wurden chemisch synthetisiert , wie 2&#39; - O - methyl RNA enthaltenden 5&#39;- und 3&#39;-Phosphate. Diese sgRNAs wurden die Auswirkungen auf die Lebensfähigkeit eines menschlichen Leukämie-Zelll...

Watch this Scientific Journal Video about TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents at JoVE.com

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

Hier präsentieren wir ein Protokoll für die Vorführung eines Heptamers-Typ sgRNA Bibliothek für potenzielle therapeutische Medikamente gegen Blutkrebs.

TRUE Gene Silencing: Screening eines Heptamer-Typ Kleiner Leitfaden RNA-Bibliothek für potentielle Krebs Therapeutika

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes ...

The overall goal of this screening is to find potential therapeutic drugs against blood cancers.Begin this procedure by selecting a subset of Heptamer sequences.The sequences can be random, or designed to target specific cellular RNAs.To target specific cellular RNAs, use an appropriate computer program to find hairpin structures resembling the T-arm in a target RNA.Select complementary seven-nucleotide sequences immediately downstream of the hairpin.Using the phosphoramidite method on the controlled pore glass, or CPG support, synthesize each of the selected Heptamers as a fully 2-prime O-methylated, 5-prime and 3-prime phosphorylated RNA with the DNA-RNA synthesizer.Leave the 5-prime terminal DMT protecting group on in the last cycle of the synthesis.After completion of the last cycle, transfer the CPG with the synthesized oligonucleotide from the column to a glass vial with a teflon-lined screw cap.Add 1mL of 29%ammonium hydroxide and incubate the tube at 55

true-gene-silencing-screening-heptamer-type-small-guide-rna-library

Research

JoVE Journal

Medicine

5.6K Aufrufe.  Niigata University of Pharmacy and Applied Life Sciences. The overall goal of this screening is to find potential therapeutic drugs against blood cancers.Begin this procedure by selecting a subset of Heptamer sequences.The sequences can be random, or designed to target specific cellular RNAs.To target specific cellular RNAs, use an appropriate computer program to find hairpin structures resembling the T-arm in a target RNA.Select complementary seven-nucleotide sequences immediately downstream of the hairpin.Using the phosphoramidite method on the co...

Serie: TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

Bronchial Thermoplasty:  A Novel Therapeutic Approach to Severe Asthma

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks. Bronchial thermoplasty is delivered by the Alair System and is performed in three outpatient procedure visits, each scheduled approximately three weeks apart. The first procedure treats the airways of the right lower lobe, the second treats the airways of the left lower lobe and the third and final procedure treats the airways in both upper lobes. After all three procedures are performed the bronchial thermoplasty treatment is complete.
Bronchial thermoplasty is performed during bronchoscopy with the patient under moderate sedation. All accessible airways distal to the mainstem bronchi between 3 and 10 mm in diameter, with the exception of the right middle lobe, are treated under bronchoscopic visualization. Contiguous and non-overlapping activations of the device are used, moving from distal to proximal along the length of the airway, and systematically from airway to airway as described previously. Although conceptually straightforward, the actual execution of bronchial thermoplasty is quite intricate and procedural duration for the treatment of a single lobe is often substantially longer than encountered during routine bronchoscopy. As such, bronchial thermoplasty should be considered a complex interventional bronchoscopy and is intended for the experienced bronchoscopist. Optimal patient management is critical in any such complex and longer duration bronchoscopic procedure. This article discusses the importance of careful patient selection, patient preparation, patient management, procedure duration, postoperative care and follow-up to ensure that bronchial thermoplasty is performed safely.
Bronchial thermoplasty is expected to complement asthma maintenance medications by providing long-lasting asthma control and improving asthma-related quality of life of patients with severe asthma. In addition, bronchial thermoplasty has been demonstrated to reduce severe exacerbations (asthma attacks) emergency rooms visits for respiratory symptoms, and time lost from work, school and other daily activities due to asthma.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks.

Bronchial Thermoplastie: einen neuen therapeutischen Ansatz bis schwerem Asthma

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled ...

Forschung

Medizin

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

Orthotopen Xenografting of Human Luciferase-Tagged malignen peripheren Nervenscheide Tumorzellen für In vivo Prüfung von Kandidaten Therapeutika

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

Abgabe therapeutischer Wirkstoffe durch intracerebroventrikuläre (ICV) und intravenöse (IV) Injektion in Mäuse

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma (glioma) and medulloblastoma. The widely used stereotactic approach only allows for the injection of a single animal at a time, is labor intensive and requires highly specialized equipment. The guide screw method, initially developed by Lal et al.,1 was developed to eliminate cumbersome stereotactic procedures. We now describe a modified guide screw approach that is rapid and exceptionally safe; both of which are critical ethical considerations. Notably, our procedure now incorporates an infusion pump that allows up to 10 animals to be simultaneously injected with tumor cells.
To demonstrate the utility of this procedure, we established human U87MG glioma cells as intracranial xenografts in mice, which were then treated with AMG102; a fully human antibody directed to HGF/scatter factor currently undergoing clinical evaluation2-5. Systemic injection of AMG102 significantly prolonged the survival of all mice with intracranial U87MG xenografts and resulted in a number of complete cures. 
This study demonstrates that the guide screw method is an inexpensive, highly reproducible approach for establishing intracranial xenografts. Furthermore, it provides a relevant physiological model for validating novel therapeutic strategies for the treatment of brain cancers.

In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.

Eine einfache Anleitung Screw Verfahren zur Intrakranielle Xenograft Studien an Mäusen

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing1, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible2,3. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. 
We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. 
Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer4,5, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

DNA-Vektor-basierte RNA-Interferenz, um Genfunktionen in Cancer Study

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

A Novel Surgical Ansatz intratracheale Verabreichung von Wirkstoffen in einem Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Screening für Melanom Modifiers mit einem Zebrafisch Autochthone Tumormodell

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

Ein orthotopen Blasenkrebs-Modell für Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

Fokus Formation: Ein Assay auf Zellbasis, um das onkogene Potential eines Gens zu bestimmen

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

Disposable Dosators for Pulmonary Insufflation of Therapeutic Agents to Small Animals

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver drugs locally and systemically. Evaluation of dry powder aerosols necessitates an efficient dispersion mechanism to maintain high concentrations in an exposure chamber or for direct endotracheal administration. While solutions exist to expose animals by passive inhalation to dry powder aerosols, most require masses of powder in large excess of the dose delivered. This precludes conducting early feasibility studies as insufficient drug is available at the research or early development stage to support the dose delivery requirements for conventional aerosol delivery systems. When designing an aerosol drug product, aerodynamic performance can relate directly to delivery efficiency and efficacy. Dispersion of powder into an aerosol requires energy input sufficient to overcome interparticulate forces, and particle engineering approaches can substantially improve aerosol performance. We have developed a dispersion system (dosator) which can aerosolize engineered dry powder aerosols efficiently for the purpose of direct pulmonary insufflation, dispersion into an exposure system or generation for analytical purposes.

During development of drugs for pulmonary delivery, it is necessary to evaluate pharmacokinetics and efficacy in an animal model. We present a method to build a disposable aerosol dispersion system from of-the-shelf components that can be used to administer intrapulmonary dry powder aerosol to rodents.

Einweg-Dosierer für Lungen Insufflation von therapeutischen Wirkstoffen zu Kleintieren

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver ...