Name: true gene silencing: screening of a heptamer-type small guide rna library for potential cancer therapeutic agents
Uploaded: 2016-06-02T14:00:00.000+00:00
Duration: 9 min 18 s
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

这项工作是由适应性和无缝技术转移计划，通过目标驱动的研发，日本科学技术振兴机构，从促进和互助公司为日本的民办学校学研究促进基金JSPS KAKENHI授权号码24300342和15H04313的支持， 。

1.一个七聚物型的构建因组库<ol><li>除非满量程库是要构造选择16,384（4 7）7-核苷酸序列的子集。 注:序列可以是无规和/或设计为靶向特定的细胞的RNA。对于后者，发现发夹结构类似的tRNA的T形臂与立即的发夹结构4,10的下游7-核苷酸序列互补的合适的计算机程序和/或视觉，和选择的序列的辅助靶RNA， 17,18。 </li><li>合成每个选定七聚体的作为完全2&#39;-O-甲基化的，5&#39;-和用DNA / RNA合成3&#39;-磷酸化的RNA。使用的可控孔度玻璃（CPG）载体的亚磷酰胺法，并留下5&#39;-末端4,4&#39;-二甲氧基三苯甲基（DMT）中的最后一个周期的保护上基（在加入5&#39;磷酸）的合成23。 注意:自定义SYnthetic RNA可从寡核苷酸的供应商也能够得到。 </li><li>最后合成循环结束后，在55℃下转移的CPG与从柱到用聚四氟乙烯衬垫螺帽玻璃小瓶中的合成的寡核苷酸中，添加氢氧化1毫升29％的硫酸铵，孵育它8小时到切割从CPG寡核苷酸和来自基地和磷酸盐除去保护基团。 </li><li>蒸发氢氧化铵用离心蒸发器在40℃和在0.1M正己基为0.2ml乙酸重溶解该寡核苷酸。 </li><li>使用聚苯乙烯 - 二乙烯基苯柱，用含10-50％乙腈/的0.1M正己基乙酸的缓冲液纯化通过反相高效液相色谱法合成的寡核苷酸。为2.0毫升，在80℃下运行柱/分钟25分钟。 </li><li>添加0.25-0.5毫升浓乙酸至分馏样品（1-2毫升），使20％的乙酸溶液，并incuba德此溶液1小时，在室温下除去DMT基团。 </li><li>在40℃干燥用离心蒸发器将样品，在氢氧化1毫升29％硫酸铵重新溶解，并在室温下孵育15分钟，该溶液以除去5&#39;磷酸的侧链。 </li><li>降低样品在40℃下的体积（1ml）中，以约0.3ml用离心蒸发器。 </li><li>透析用分子量的透析管截止1000在4℃过夜对蒸馏水整个浓缩样品（500毫升）。 </li><li>从透析管用吸管转移透析的样品，以1.5毫升管中，在40℃下用离心蒸发器干燥它，并随后在水中换注射级水重新溶解，其容积应小到足以使一个大于100微米的解决方案。 </li><li>测量的RNA样品的光学密度在用分光光度计260nm处，并调整其浓度至100通过加入水换注射级水μM。 -20°C使用前存放在下面的RNA溶液。 </li></ol> 2.筛选因组文库的细胞活力<ol><li>制备10 3个细胞/ 99微升/孔（ 例如，HL60和RPMI-8226）上在补充有10％胎牛血清和1％青霉素-链霉素溶液的RPMI-1640培养基的96孔培养皿中。准备包含媒介只为背景扣除井。消除边缘效果，添加无菌水到清空围绕样品井孔中。 </li><li>加入1μl在水中溶解换注射级水至各孔七聚物型因组（100μM）的。测定每个因组一式三份。在3天，5％CO 2加湿培养箱中孵育该板在37℃。 </li><li>添加市售的WST-8溶液以每孔的适当体积。孵育在37℃的板中进行1至4小时，以同一培养箱。 </li><li>测量吸光度吨，酶标仪450纳米。 注:在大多数情况下，吸光率和存活细胞数之间的线性显示，除非吸光度值超过1.0要维持。 </li></ol> 3.有效sgRNAs评价流式细胞仪<ol><li>制备10 3个细胞/在补充有10％胎牛血清和1％青霉素-链霉素溶液的RPMI-1640培养基的96孔培养皿99微升/孔（ 例如，HL60）。制备至少10井进行测试1因组。消除边缘效果，添加无菌水到清空围绕样品井孔中。 </li><li>加入1μl在水中溶解换注射级水至各孔七聚物型因组（100μM）的。在3天，5％CO 2加湿培养箱中孵育该板在37℃。 </li><li>收集具有相同的因组从至少10个孔处理到一个5毫升试管用移液器细胞。旋管5分钟在离心机中，在300 XG，弃媒体。 </li><li> 2毫升1×磷酸盐缓冲盐水（PBS）添加到管中，并重新悬浮细胞。旋管5分钟在离心机中以300 xg离心，并弃去PBS。添加150微升1×膜联蛋白V结合缓冲液至管中，并重新悬浮细胞。 </li><li>添加2微升藻红蛋白的（​​PE）缀合的膜联蛋白V溶液和1μl7氨基放线菌素D（7AAD）溶液于管，将它们混合均匀，并孵育管在黑暗中在室温下15分钟。分析用流式细胞仪24的流动的样品。 </li></ol>

<ol>
	<li>Scherer, L., Rossi, J. J., Morris, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+RNA-mediated+control+of+gene+expression:+Options+and+designs.">Therapeutic potential of RNA-mediated control of gene expression: Options and designs.</a> RNA and the Regulation of Gene Expression: A hidden layer of complexity. , 201-226 (2008).</li><li>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=Conversion+of+mammalian+tRNA+3&#8242;+processing+endoribonuclease+to+four-base-recognizing+RNA+cutters.">Conversion of mammalian tRNA 3&#8242; processing endoribonuclease to four-base-recognizing RNA cutters.</a> Nucleic Acids Res. 23, 3642-3647 (1995).</li><li>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=Specific+cleavage+of+target+RNAs+from+HIV-1+with+5&#8242;+half+tRNA+by+mammalian+tRNA+3&#8242;+processing+endoribonuclease.">Specific cleavage of target RNAs from HIV-1 with 5&#8242; half tRNA by mammalian tRNA 3&#8242; processing endoribonuclease.</a> RNA. 2, 523-534 (1996).</li><li>Nashimoto, M., Geary, S., Tamura, M., Kasper, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+heptamers+that+directs+RNA+cleavage+by+mammalian+tRNA+3&#8242;+processing+endoribonuclease.">RNA heptamers that directs RNA cleavage by mammalian tRNA 3&#8242; processing endoribonuclease.</a> Nucleic Acids Res. 26, 2565-2571 (1998).</li><li>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=Anomalous+RNA+substrates+for+mammalian+tRNA+3&#8242;+processing+endoribonuclease.">Anomalous RNA substrates for mammalian tRNA 3&#8242; processing endoribonuclease.</a> FEBS Lett. 472, 179-186 (2000).</li><li>Takaku, H., Minagawa, A., Takagi, 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=A+candidate+prostate+cancer+susceptibility+gene+encodes+tRNA+3&#8242;+processing+endoribonuclease.">A candidate prostate cancer susceptibility gene encodes tRNA 3&#8242; processing endoribonuclease.</a> Nucleic Acids Res. 31, 2272-2278 (2003).</li><li>Takaku, H., Minagawa, A., Takagi, 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=The+N-terminal+half-domain+of+the+long+form+of+tRNase+Z+is+required+for+the+RNase+65+activity.">The N-terminal half-domain of the long form of tRNase Z is required for the RNase 65 activity.</a> Nucleic Acids Res. 32, 4429-4438 (2004).</li><li>Takaku, H., Minagawa, A., Takagi, 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=A+novel+four-base-recognizing+RNA+cutter+that+can+remove+the+single+3&#8242;+terminal+nucleotides+from+RNA+molecules.">A novel four-base-recognizing RNA cutter that can remove the single 3&#8242; terminal nucleotides from RNA molecules.</a> Nucleic Acids Res. 32, e91 (2004).</li><li>Shibata, H. S., Takaku, H., Takagi, 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=The+T+loop+structure+is+dispensable+for+substrate+recognition+by+tRNase+ZL.">The T loop structure is dispensable for substrate recognition by tRNase ZL.</a> J. Biol. 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...

真基因沉默（TR核苷酸酶Ž 信用证后称为- U tilizingËfficacious基因沉默）是RNA指导的基因沉默技术1中的一个。这种技术已经开发了基于tRNasežL（或3&#39;tRNase），tRNA的3&#39;加工核酸内切酶的特性:它可以通过识别切割的人造小导RNA（因组）的指导下，在任何预期的网站上的任何靶RNA 9 -靶RNA和因组2之间形成酰tRNA样前或微预tRNA的状结构。因组，这通常是7-31个核苷酸长，被分类为四组，5&#39;-半酰tRNA，七聚体RNA，14-nt的线性RNA和钩的RNA。 15 -我们已经通过引入各种人为设计sgRNAs入活细胞10展示为TRUE基因沉默的效果。我们还表明，sgRNAs可以被细胞的Wi被吸收thout任何转染试剂，可以下调它们的靶RNA水平和/或在人类癌细胞16诱导细胞凋亡- 18。 TRUE基因沉默似乎就通过tRNasežL和自然sgRNAs 19细胞内和细胞间的基因调控网络系统的工作- 21。 我们已经构建了含156七聚物型sgRNAs一个因组文库以寻找潜在的治疗七聚物型sgRNAs对于血癌22。我们筛选其对人骨髓瘤和白血病细胞的生存力的影响，并发现其中20能有效地诱导凋亡的肿瘤细胞株中的至少一种。和他们的4已经显示减少在小鼠异种移植实验肿瘤的生长速率。 在这里，我们提出了一个协议为七聚物型因组库潜在的治疗药物抗血癌的筛查。该协议包括如何构造因组文库，如何评估细胞生存力，以及如何进一步通过流式细胞术评估的有效sgRNAs每因组的效果。可以根据常规方法17,22虽然这些未在此方案包括进行分析因组的细胞靶RNA和有效sgRNAs评价在小鼠异种移植模型。

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

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

真基因沉默（TR核苷酸酶Ž 信用证后称为- U tilizingËfficacious基因沉默）是RNA指导的基因沉默技术，它利用的人造小导的RNA（因组）引导的tRNA 3&#39;加工内切核糖核酸酶，tRNasežL的一个，以识别靶RNA。 sgRNAs可以采取由细胞没有任何转染试剂，可以下调它们的靶RNA水平和/或在人类癌细胞中诱导细胞凋亡。我们筛选含有156对人骨髓瘤和白血病细胞的生存力的影响七聚物型sgRNAs一个因组文库，并发现其中20能有效地诱导凋亡的肿瘤细胞株中的至少一种。在这里，我们提出了一个协议为七聚物型因组库潜在的治疗药物抗血癌的筛查。该协议包括如何构造因组文库，如何评估细胞生存力，以及各因组的效果如何复rther评估通过流式细胞仪的有效sgRNAs。约2,000命中预计将通过筛选的16,384七聚体构成的全规模因组文库来获得。

六个七聚物型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），和5&#39;-CACCAGC-3&#39;（H13260）作为含有5&#39;-和3&#39;-磷酸酯2&#39;-O-甲基RNA的化学合成。 这些sgRNAs检查用于在人白血病细胞系，HL60和人骨髓瘤细胞系，RPMI-8226的存活力的?...

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

在这里，我们提出了一个协议为七聚物型因组库潜在的治疗药物抗血癌的筛查。

TRUE基因沉默:一个七聚物型小导RNA库的筛选潜在的癌症治疗药

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

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

5.6K 次观看.  Niigata University of Pharmacy and Applied Life Sciences. 在这里，我们提出了一个协议为七聚物型因组库潜在的治疗药物抗血癌的筛查。 

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

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

全基因组的 rna 干扰筛查以确定调节溶瘤病毒治疗的宿主因素

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

科研

癌症研究

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

A CRISPR/sgRNA library has been applied to interrogating protein-coding genes.However, the feasibility of a sgRNA library to uncover the function of a CTCF boundary in gene regulation remains unexplored. Here, we describe a HOX loci specific sgRNA library to elucidate the function of CTCF boundaries in HOX loci.

公司重点调查的社区图书馆筛选确定关键 CTCF 边界

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements ...

遗传学

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

CRISPR-Cas9 technology provides an efficient method to precisely edit the mammalian genome in any cell type and represents a novel means to perform genome-wide genetic screens. A detailed protocol discussing the steps required for the successful performance of pooled genome-wide CRISPR-Cas9 screens is provided here.

哺乳动物细胞中基于CRISPR的聚集基因屏幕

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian ...

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

使用阵列库的中等通量筛选和基于双 Luciferase 的分录报告器识别转录因子稳压器

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

医学

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve survival in patients with acute myeloid leukemia (AML). A small fraction of leukemic cells that survive chemotherapy have a poised epigenetic state to tolerate chemotherapeutic insult. Further exposure to chemotherapy allows these drug persister cells to attain a fixed epigenetic state, which leads to altered gene expression, resulting in the proliferation of these drug-resistant populations and eventually relapse or refractory disease. Therefore, identifying epigenetic modulations that necessitate the survival of drug-resistant leukemic cells is critical. We detail a protocol to identify epigenetic modulators that mediate resistance to the nucleoside analog cytarabine (AraC) using pooled shRNA library screening in an acquired cytarabine-resistant AML cell line. The library consists of 5,485 shRNA constructs targeting 407 human epigenetic factors, which allows high-throughput epigenetic factor screening.

High-throughput RNA interference (RNAi) screening using a pool of lentiviral shRNAs can be a tool to detect therapeutically relevant synthetic lethal targets in malignancies. We provide a pooled shRNA screening approach to investigate the epigenetic effectors in acute myeloid leukemia (AML).

合并的shRNA文库筛选，以确定调节耐药性表型的因素

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve ...

基因组规模筛选，用于识别和比较肿瘤中的治疗靶点

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. 
In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. 
This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

作者聚焦:通过基因组规模 shRNA 筛选寻找恶性周围神经鞘瘤的新治疗靶点 

We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type.

遗传分析和基因组规模的辍学筛选以确定恶性周围神经鞘瘤小鼠模型中的治疗靶点

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome ...

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

TRUE基因沉默:一个七聚物型小导RNA库的筛选潜在的癌症治疗药

摘要

探索更多视频

此视频中的章节

全基因组的 rna 干扰筛查以确定调节溶瘤病毒治疗的宿主因素

公司重点调查的社区图书馆筛选确定关键 CTCF 边界

哺乳动物细胞中基于CRISPR的聚集基因屏幕

使用阵列库的中等通量筛选和基于双 Luciferase 的分录报告器识别转录因子稳压器

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

合并的shRNA文库筛选，以确定调节耐药性表型的因素

遗传分析和基因组规模的辍学筛选以确定恶性周围神经鞘瘤小鼠模型中的治疗靶点

遗传分析和基因组规模的辍学筛选以确定恶性周围神经鞘瘤小鼠模型中的治疗靶点

遗传分析和基因组规模的辍学筛选以确定恶性周围神经鞘瘤小鼠模型中的治疗靶点

miRNA的目标使用的任务目标ID库的全基因组屏幕