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

Este trabalho foi apoiado pelo Programa de Transferência adaptável e Seamless Tecnologia através de I &amp; D, o Japão Agência de Ciência e Tecnologia, Fundo de Promoção de pesquisa da ciência da Corporação de Promoção e Ajuda Mútua para Escolas Particulares do Japão Target-driven, e os números JSPS KAKENHI Grant 24300342 e 15H04313 .

1. Construção de um tipo de heptâmero sgRNA Biblioteca <ol><li> Selecionar um subconjunto dos 16.384 (4 7) sequências de 7 nt a menos que a biblioteca em grande escala deve ser construída. NOTA: As sequências podem ser aleatórios e / ou projetados para atingir RNAs celulares específicos. Para este último, encontrar estruturas em gancho de cabelo que se assemelha à do t-braço de ARNt num ARN alvo com o auxílio de um programa adequado de computador e / ou visualmente, e seleccione sequênci...

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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...

Silenciamento do gene VERDADEIRO (denominado após tR Nase Z L - L e tilizing fficacious silenciamento do gene) é uma das tecnologias de silenciamento do gene dirigida por RNA 1. Esta tecnologia foi desenvolvida com base nas propriedades de tRNase Z L (ou 3 &#39;tRNase), ARNt 3&#39; endorribonuclease processamento: ela pode clivar qualquer ARN alvo em qualquer sítio esperado sob a direcção de um pequeno ARN guias artificiais (sgRNA) através do reconhecimento de uma pré-ARNt ou de tipo micro-pré-ARNt-como a estrutura formada entre o RNA alvo e a sgRNA 2-9. sgRNA, que normalmente é 7-31 nt de comprimento, é categorizada em quatro grupos, 5&#39;-meia-ARNt, ARN heptâmero, ARN linear de 14 nt, e de ARN de gancho. Temos demonstrado a eficácia de silenciamento de genes VERDADEIRO através da introdução de vários sgRNAs artificialmente projetados em células vivas 10-15. Também mostrámos que sgRNAs pode ser absorvida pelas células Without quaisquer reagentes de transfecção e pode regular negativamente os seus níveis de RNA alvo e / ou induzir a apoptose em células cancerosas humanas 16 - 18. Silenciamento de genes VERDADEIRO aparece para trabalhar no intracelular e sistema de rede de transcrição intercelular via tRNase Z L e sgRNAs naturais 19 - 21. Construímos uma biblioteca sgRNA contendo 156 do tipo heptâmero sgRNAs para procurar potenciais sgRNAs do tipo heptâmero terapêuticas para cancros do sangue 22. Temos rastreados para que o efeito sobre a viabilidade de células de mieloma e de leucemia humana, e descobriram que 20 deles pode eficientemente induzir a apoptose em pelo menos uma das linhas celulares de cancro. E quatro deles foram mostrados para reduzir as taxas de crescimento de tumor em experiências de xenoenxerto de ratinho. Aqui apresentamos um protocolo para o rastreio de uma biblioteca sgRNA tipo heptâmero para os potenciais drogas terapêuticas contra o câncer no sangue. O protocolo inclui comopara construir a biblioteca sgRNA, a forma de avaliar o efeito de cada sgRNA sobre a viabilidade celular, e a forma de avaliar ainda mais os sgRNAs eficazes por citometria de fluxo. Análise para ARN alvo celular da sgRNA e avaliação de sgRNAs eficazes em modelos de xenoenxerto de ratinho pode ser realizada de acordo com métodos convencionais 17,22, embora estes não estão incluídas no presente protocolo.

<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

Silenciamento do gene VERDADEIRO (denominado após tR Nase Z L - L tilizing e fficacious silenciamento do gene) é um do gene dirigida por ARN de silenciamento tecnologias, que utiliza uma pequena guia de ARN artificial (sgRNA) para guiar endorribonuclease processamento ARNt 3 &#39;, tRNase Z L , para reconhecer um alvo de ARN. sgRNAs podem ser absorvidos pelas células sem quaisquer reagentes de transfecção e pode regular negativamente os seus níveis de RNA alvo e / ou induzir a apoptose em células cancerosas humanas. Temos rastreada uma biblioteca sgRNA contendo 156 sgRNAs do tipo heptâmero para o efeito sobre a viabilidade de células de mieloma e de leucemia humana, e descobriram que 20 deles pode eficientemente induzir a apoptose em pelo menos uma das linhas celulares de cancro. Aqui apresentamos um protocolo para o rastreio de uma biblioteca sgRNA tipo heptâmero para os potenciais drogas terapêuticas contra o câncer no sangue. O protocolo inclui a forma de construir a biblioteca sgRNA, a forma de avaliar o efeito de cada sgRNA sobre a viabilidade celular, e a forma de further avaliar os sgRNAs eficazes por citometria de fluxo. Cerca de 2000 acessos seria esperado para ser obtido por rastreio da biblioteca em grande escala sgRNA composto de 16.384 heptamers.

Os seis tipo heptâmero 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) foram sintetizados quimicamente como 2&#39;-O-metil ARN contendo 5&#39;- e 3&#39;-fosfatos. Estes sgRNAs foram examinados quanto aos efeitos sobre a viabilidade de uma linha celular de leucemia human...

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

Aqui apresentamos um protocolo para o rastreio de uma biblioteca sgRNA tipo heptâmero para os potenciais drogas terapêuticas contra o câncer no sangue.

VERDADEIRO silenciamento gênico: Triagem de um heptâmero tipo Guia de pequeno RNA Biblioteca de agentes terapêuticos potenciais Câncer

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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