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

Dit werk werd ondersteund door flexibele en naadloze Technology Transfer Program via Target-gedreven R &amp; D, Japan Science and Technology Agency, de Science Research Stimuleringsfonds voor de actie en de Mutual Aid Corporation for Private Schools of Japan, en de JSPS KAKENHI Grant Numbers 24300342 en 15H04313 .

1. Bouw van een heptameer-type sgRNA Library <ol><li> Selecteer een subset van de 16.384 (4 7) 7-nt sequenties tenzij de full-scale bibliotheek te construeren. LET OP: De sequenties kunnen willekeurig en / of om specifieke cellulaire RNA&#39;s doelwit. Voor dit laatste voorbeeld haarspeldstructuren lijkt de T-arm van tRNA in een doel-RNA met behulp van een geschikt computerprogramma en / of visueel en selecteer sequenties complementair aan 7-nt sequenties onmiddellijk stroomafwaarts van de haarspelds...

<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 genuitschakeling (genoemd na tR Nase ZL - u tilizing e fficacious genuitschakeling) is een van de RNA-gerichte gene silencing technologie 1. Deze technologie werd ontwikkeld op basis van eigenschappen van tRNase ZL (of 3 &#39;tRNase), tRNA 3&#39; verwerking endoribonuclease: het kan elke doel-RNA op een verwachte plaats splitsen onder leiding van een kunstmatige geleidepen RNA (sgRNA) door het herkennen pre-tRNA-achtige of micro-pre-tRNA-achtige structuur gevormd tussen het doelwit-RNA en sgRNA 2-9. sgRNA, dat gewoonlijk 7-31 nt lang is onderverdeeld in vier groepen, 5&#39;-half-tRNA, heptameer RNA, 14-nt lineair RNA en RNA haak. We hebben de effectiviteit van TRUE gene silencing aangetoond door de invoering van verschillende kunstmatig ontworpen sgRNAs in levende cellen 10-15. We hebben ook aangetoond dat sgRNAs kan worden opgenomen door cellen without elke transfectiereagentia en kunnen hun doelwit RNA-spiegels downregulate en / of apoptose in menselijke kankercellen 16-18. TRUE genuitschakeling lijkt te werken op de intracellulaire en intercellulaire genregulerend netwerksysteem via tRNase ZL en natuurlijke sgRNAs 19-21. We hebben een sgRNA bibliotheek met 156 heptameer-type sgRNAs geconstrueerd om te zoeken naar potentiële therapeutische heptameer-type sgRNAs voor bloedkanker 22. We hebben het gescreend op het effect op de levensvatbaarheid van humane myeloma en leukemie cellen en vonden dat 20 ze efficiënt apoptose in ten minste één van de kanker cellijnen kan induceren. En 4 ervan is aangetoond dat tumorgroeisnelheden in muis xenograft experimenten verminderen. Hier presenteren we een protocol voor het screenen van een heptameer-type sgRNA bibliotheek voor potentiële therapeutische geneesmiddelen tegen bloedkanker. Het protocol bevat hoehet sgRNA construeren, hoe het effect van elke sgRNA op cellevensvatbaarheid en hoe de effectieve sgRNAs verdere beoordeling door flowcytometrie beoordelen. Analyse voor sgRNA de cellulaire doelwit RNA en evaluatie van effectieve sgRNAs in muizen xenograft modellen kan worden uitgevoerd volgens conventionele werkwijzen 17,22, hoewel deze niet in dit protocol.

<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 genuitschakeling (genoemd na tR Nase ZL - u tilizing e fficacious genuitschakeling) is een van de RNA-gerichte gen silencing technieken, die een kunstmatige geleidepen RNA (sgRNA) ingezet om tRNA 3 &#39;verwerking endoribonuclease, tRNase ZL gids , een doel-RNA te herkennen. sgRNAs kunnen door cellen worden zonder transfectie reagentia en hun doel-RNA niveaus downreguleren en / of apoptose in humane kankercellen. We hebben een sgRNA bibliotheek met 156 heptameer type sgRNAs voor het effect op de levensvatbaarheid van humane myeloma en leukemie cellen onderzocht en vonden dat 20 ze efficiënt apoptose in ten minste één van de kanker cellijnen kan induceren. Hier presenteren we een protocol voor het screenen van een heptameer-type sgRNA bibliotheek voor potentiële therapeutische geneesmiddelen tegen bloedkanker. Het protocol bevat hoe de sgRNA bibliotheek te construeren, hoe het effect van elke sgRNA op de levensvatbaarheid van de cellen, en te beoordelen hoe om te further evalueren effectieve sgRNAs met flowcytometrie. Rond 2000 treffers zou naar verwachting worden verkregen door het screenen van de full-scale sgRNA bibliotheek bestaat uit 16.384 heptameren.

De zes heptameer type 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) en 5&#39;-CACCAGC-3&#39; (H13260) werden chemisch gesynthetiseerd als 2&#39;-O-methyl RNA-bevattende 5&#39;- en 3&#39;-fosfaten. Deze sgRNAs werden onderzocht op de effecten op de levensvatbaarheid van een menselijke leukemie cellijn, ...

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 presenteren we een protocol voor het screenen van een heptameer-type sgRNA bibliotheek voor potentiële therapeutische geneesmiddelen tegen bloedkanker.

TRUE Gene Silencing: Screening van een heptameer-type Kleine Gids RNA bibliotheek voor potentiële Cancer Therapeutische Agents

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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