Name: true gene silencing: screening of a heptamer-type small guide rna library for potential cancer therapeutic agents
Uploaded: 2016-06-02T14:00:00
Description: 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

Questo lavoro è stato sostenuto da adattabile e senza soluzione di continuità la tecnologia dei programmi di trasferimento attraverso la R &amp; S, il Giappone Scienza e tecnologia Agenzia, il Fondo di promozione di ricerca di scienza dalla Promozione e l&#39;aiuto reciproco Corporation per Private Schools of Japan target-driven, e il numero JSPS KAKENHI sovvenzione 24.300.342 e 15H04313 .

1. Costruzione di un Heptamer tipo sgRNA libreria <ol><li> Selezionare un sottoinsieme di 16.384 (4) 7 sequenze 7-NT a meno che la libreria di fondo scala deve essere costruito. NOTA: Le sequenze possono essere casuale e / o progettati per indirizzare RNA cellulari specifici. Per questi ultimi, per strutture forcella simile al T-braccio tRNA in un RNA bersaglio con l&#39;ausilio di un idoneo programma di computer e / o visivo, e selezionare le sequenze complementari a sequenze 7-nt immediatamente a v...

<ol>
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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>

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 silenziamento genico (denominato dopo tR Nase Z L - u tilizing e fficacious silenziamento genico) è una delle tecnologie silenziamento genico RNA-diretto 1. Questa tecnologia è stata sviluppata sulla base di proprietà di tRNase Z L (o 3 &#39;tRNase), tRNA 3&#39; endoribonuclease lavorazione: può fendere qualsiasi RNA bersaglio in qualsiasi luogo previsto sotto la direzione di una guida piccoli RNA artificiale (sgRNA) riconoscendo un pre-tRNA-like o micro-pre-tRNA-come struttura formata tra l&#39;RNA bersaglio e la sgRNA 2-9. sgRNA, che di solito è 7-31 nt di lunghezza, è classificato in quattro gruppi, 5&#39;-mezzo-tRNA, RNA heptamer, 14-nt RNA lineare e RNA gancio. Abbiamo dimostrato l&#39;efficacia di TRUE silenziamento genico con l&#39;introduzione di vari sgRNAs artificialmente progettati in cellule viventi 10-15. Abbiamo anche dimostrato che sgRNAs possono essere presi dalle cellule without eventuali reagenti di trasfezione e può downregulate i loro livelli di RNA di riferimento e / o indurre l&#39;apoptosi nelle cellule tumorali umane 16 - 18. TRUE silenziamento genico sembra funzionare sul intracellulare e sistema di rete di regolazione genica intercellulare tramite tRNase Z L e sgRNAs naturali 19 - 21. Abbiamo costruito una libreria sgRNA contenente 156 heptamer tipo sgRNAs per la ricerca di potenziali sgRNAs heptamer tipo terapeutici per i tumori del sangue 22. Abbiamo proiettato per l&#39;effetto sulla vitalità delle cellule di mieloma e leucemia umana, e trovato che 20 di essi può efficientemente indurre apoptosi in almeno una delle linee di cellule di cancro. E 4 di loro hanno dimostrato di ridurre i tassi di crescita del tumore negli esperimenti di xenotrapianto del mouse. Qui vi presentiamo un protocollo per lo screening di una libreria sgRNA heptamer-tipo per farmaci terapeutici potenziali contro i tumori del sangue. Il protocollo comprende comeper costruire la biblioteca sgRNA, come valutare l&#39;effetto di ogni sgRNA sulla vitalità delle cellule, e come valutare ulteriormente le sgRNAs efficaci mediante citometria di flusso. Analisi per l&#39;RNA bersaglio cellulare di sgRNA e la valutazione delle sgRNAs efficaci in modelli di xenotrapianto mouse possono essere eseguite secondo metodi convenzionali 17,22, anche se questi non sono inclusi in questo protocollo.

<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 silenziamento genico (chiamato dopo tR Nase Z L - u tilizing e fficacious silenziamento genico) è uno dei geni RNA-regia tacere tecnologie, che utilizza un artificiale piccola guida RNA (sgRNA) per guidare tRNA 3 &#39;endoribonuclease elaborazione, tRNase Z L , di riconoscere un RNA bersaglio. sgRNAs possono essere presi dalle cellule senza reagenti di trasfezione e possono downregulate i loro livelli di RNA di riferimento e / o indurre l&#39;apoptosi nelle cellule tumorali umane. Abbiamo verificato una libreria sgRNA contenente 156 heptamer tipo sgRNAs per l&#39;effetto sulla vitalità delle cellule di mieloma e leucemia umana, e trovato che 20 di essi può efficientemente indurre apoptosi in almeno una delle linee di cellule di cancro. Qui vi presentiamo un protocollo per lo screening di una libreria sgRNA heptamer-tipo per farmaci terapeutici potenziali contro i tumori del sangue. Il protocollo comprende come costruire la libreria sgRNA, come valutare l&#39;effetto di ogni sgRNA sulla vitalità delle cellule, e come further valutare le sgRNAs efficaci mediante citometria di flusso. Circa 2.000 colpi ci si aspetterebbe da versare lo screening della libreria sgRNA su vasta scala composta da 16.384 heptamers.

I sei heptamer tipo 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), e 5&#39;-CACCAGC-3&#39; (H13260) sono stati sintetizzati chimicamente come 2&#39;- O metil RNA contenente 5&#39;- e 3&#39;-fosfati. Questi sgRNAs sono stati esaminati per gli effetti sulla vitalità di una linea cellulare di leucemia um...

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

Qui vi presentiamo un protocollo per lo screening di una libreria sgRNA heptamer-tipo per farmaci terapeutici potenziali contro i tumori del sangue.

TRUE silenziamento genico: Proiezione di un Heptamer di tipo piccolo RNA guida Library per potenziali agenti terapeutici cancro

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

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