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

Detta arbete stöddes av Anpassningsbar och Seamless Technology Transfer Program genom målstyrt FoU, Japan Science and Technology Agency, Science Research Promotion Fund från kampanjen och Mutual Aid Corporation för Privata skolor i Japan, och JSPS KAKENHI Grant Numbers 24300342 och 15H04313 .

1. Konstruktion av en heptameren-typ sgRNA Library <ol><li> Välj en delmängd av de 16384 (4 7) 7-nt-sekvenser inte fullskale biblioteket ska byggas. OBS: Sekvenserna kan vara slumpmässigt och / eller utformad för att inrikta sig på specifika cellulära RNA. För det senare hitta hårnålsstrukturer som liknar T-arm tRNA i en mål-RNA med hjälp av ett lämpligt datorprogram och / eller visuellt och välj sekvenser komplementära till 7-nt-sekvenser omedelbart nedströms av hårnålsstrukturerna ...

<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 geners uttryck (benämnd efter tR Nase Zl - u tilizing e fficacious geners uttryck) är en av de RNA-styrda teknik geners uttryck 1. Denna teknik har utvecklats baserat på egenskaper hos tRNase Z L (eller 3 &#39;tRNase), tRNA 3 &quot;behandling endoribonukleas: det kan klyva alla mål-RNA vid eventuellt förväntat plats under ledning av en konstgjord liten guide-RNA (sgRNA) genom att erkänna en pre-tRNA-liknande eller mikro-pre-tRNA-liknande struktur som bildas mellan mål-RNA och sgRNA 2-9. sgRNA, som vanligtvis är 7-31 nt i längd, kategoriseras i fyra grupper, 5&#39;-halv-tRNA, heptamer RNA, 14-nt linjär RNA, och krok-RNA. Vi har visat att effekten av TRUE geners uttryck genom att införa olika artificiellt utformade sgRNAs i levande celler 10-15. Vi har också visat att sgRNAs kan tas upp av celler without eventuella transfektionsreagens och kan nedreglera deras mål-RNA-nivåer och / eller inducera apoptos i humana cancerceller 16 - 18. TRUE geners uttryck verkar fungera på den intracellulära och inter genreglerande nätverkssystem via tRNase Zl och naturliga sgRNAs 19 - 21. Vi har konstruerat en sgRNA bibliotek innehållande 156 heptamer typ sgRNAs att söka efter potentiella terapeutiska heptamer-typ sgRNAs för blodcancer 22. Vi har screenas det för effekten på viabiliteten hos humana myelom och leukemiceller, och fann att 20 av dem på ett effektivt sätt kan inducera apoptos i åtminstone en av de cancercellinjer. Och fyra av dem har visat sig minska tumörtillväxttakt i mus xenograft experiment. Här presenterar vi ett protokoll för screening av en heptamer-typ sgRNA bibliotek för potentiella terapeutiska läkemedel mot blodcancer. Protokollet omfattar huratt konstruera sgRNA biblioteket, hur man bedöma effekten av varje sgRNA på cellviabilitet, och hur man ytterligare utvärdera effektiva sgRNAs genom flödescytometri. Analys för sgRNA cellulära mål RNA och utvärdering av effektiva sgRNAs i mus xenograft-modeller kan utföras i enlighet med konventionella metoder 17,22, även om dessa inte ingår i detta protokoll.

<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 geners uttryck (benämnd efter tR Nase Zl - u tilizing e fficacious geners uttryck) är en av RNA-riktad geners uttryck teknik, som utnyttjar en artificiell liten guide-RNA (sgRNA) för att styra tRNA 3 &quot;behandling endoribonukleas, tRNase Zl att erkänna en mål-RNA. sgRNAs kan tas upp av celler utan några transfektionsreagens och kan nedreglera deras mål-RNA-nivåer och / eller inducera apoptos i humana cancerceller. Vi har screened ett sgRNA bibliotek innehållande 156 heptamer-typ sgRNAs för effekten på viabiliteten hos humana myelom och leukemiceller, och fann att 20 av dem på ett effektivt sätt kan inducera apoptos i åtminstone en av de cancercellinjer. Här presenterar vi ett protokoll för screening av en heptamer-typ sgRNA bibliotek för potentiella terapeutiska läkemedel mot blodcancer. Protokollet omfattar hur man skall konstruera sgRNA biblioteket, hur man bedöma effekten av varje sgRNA på cellviabilitet, och hur man further utvärdera de effektiva sgRNAs genom flödescytometri. Omkring 2000 träffar förväntas erhållas genom screening av fullskalig sgRNA bibliotek bestående av 16384 heptamerer.

De sex 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), och 5&#39;-CACCAGC-3&#39; (H13260) syntetiserades kemiskt som 2&#39;-O-metyl-RNA innehållande 5&#39;- och 3&#39;-fosfater. granskat dessa sgRNAs var för effekterna på lönsamheten för en human leukemi cellinje HL60 och en human myel...

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

Här presenterar vi ett protokoll för screening av en heptamer-typ sgRNA bibliotek för potentiella terapeutiska läkemedel mot blodcancer.

TRUE geners: Screening av en heptameren-typ liten guide RNA bibliotek för potentiella cancer terapeutiska medel

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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