Name: genome-wide rnai screening to identify host factors that modulate oncolytic virus therapy
Uploaded: 2018-04-03T13:00:00
Description: Watch this Scientific Journal Video about Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy at JoVE.com

这项工作得到了安大略省癌症研究研究所、加拿大创新基金会、渥太华区域癌症基金会和特里福克斯研究所的资助。K.J.A. 得到了加拿大 Vanier 研究生奖学金、加拿大卫生研究所硕士奖和安大略省研究生奖学金的支持。

1. 高通量 siRNA 筛选试验的开发和验证
注: 对于所有实验, 请使用适合每个单元格线的 Hepes 缓冲生长介质。有关从分析优化到第三级验证的工作流概述, 请参见图 1 。
<ol><li>最佳转染条件的确定
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<li>选择肿瘤细胞系或理想的多肿瘤细胞系以优化转染条件 (例如786 O、NCI-H226、SF268、U373、OVCAR-8、U20S; 有关单元格线选择的更多内容, 请参...

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	<li>Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+have+RNAi+screens+taught+us+about+viral-host+interactions?.">What have RNAi screens taught us about viral-host interactions?.</a> Curr. Opin. Microbiol. 12 (4), 446-452 (2009).</li><li>Panda, D., Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+genomic+screening:+elucidating+virus-host+interactions.">Cell-based genomic screening: elucidating virus-host interactions.</a> Curr. Opin. Virol. 2 (6), 784-792 (2012).</li><li>Houzet, L., Jeang, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+screening+using+RNA+interference+to+study+host+factors+in+viral+replication+and+pathogenesis.">Genome-wide screening using RNA interference to study host factors in viral replication and pathogenesis.</a> Exp. Biol. Med. 236 (8), 962-967 (2011).</li><li>Friedel, C. C., Haas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-host+interactomes+and+global+models+of+virus-infected+cells.">Virus-host interactomes and global models of virus-infected cells.</a> Trends Microbiol. 19 (10), 501-508 (2011).</li><li>Gao, S., Yang, C., 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=Applications+of+RNA+interference+high-throughput+screening+technology+in+cancer+biology+and+virology.">Applications of RNA interference high-throughput screening technology in cancer biology and virology.</a> Protein Cell. 5 (11), 805-815 (2014).</li><li>Kilcher, S., Mercer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next+generation+approaches+to+study+virus+entry+and+infection.">Next generation approaches to study virus entry and infection.</a> Curr. Opin. Virol. 4, 8-14 (2014).</li><li>Ramage, H., Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-Host+Interactions:+From+unbiased+genetic+screens+to+function.">Virus-Host Interactions: From unbiased genetic screens to function.</a> Annu. Rev. Virol. 2 (1), 497-524 (2015).</li><li>Mercer, J., Snijder, B., 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=RNAi+screening+reveals+proteasome-+and+Cullin3-dependent+stages+in+vaccinia+virus+infection.">RNAi screening reveals proteasome- and Cullin3-dependent stages in vaccinia virus infection.</a> Cell Rep. 2 (4), 1036-1047 (2012).</li><li>Sivan, G., Martin, S. E., 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+genome-wide+RNAi+screen+reveals+a+role+for+nuclear+pore+proteins+in+poxvirus+morphogenesis.">Human genome-wide RNAi screen reveals a role for nuclear pore proteins in poxvirus morphogenesis.</a> Proc. Natl. Acad. Sci. U.S.A. 110 (9), 3519-3524 (2013).</li><li>Beard, P. M., Griffiths, S. J., 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+loss+of+function+analysis+of+host+factors+Influencing+vaccinia+virus+replication+by+RNA+Interference.">A loss of function analysis of host factors Influencing vaccinia virus replication by RNA Interference.</a> PLoS ONE. 9 (6), e98431 (2014).</li><li>Teferi, W. M., Dodd, K., Maranchuk, R., Favis, N., Evans, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+whole-genome+RNA+interference+screen+for+human+cell+factors+affecting+myxoma+virus+replication.">A whole-genome RNA interference screen for human cell factors affecting myxoma virus replication.</a> J. Virol. 87 (8), 4623-4641 (2013).</li><li>Workenhe, S. T., Ketela, T., Moffat, J., Cuddington, B. P., Mossman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+lentiviral+shRNA+screen+identifies+serine/arginine-rich+splicing+factor+2+as+a+determinant+of+oncolytic+virus+activity+in+breast+cancer+cells.">Genome-wide lentiviral shRNA screen identifies serine/arginine-rich splicing factor 2 as a determinant of oncolytic virus activity in breast cancer cells.</a> Oncogene. 35 (19), 2465-2477 (2015).</li><li>Panda, D., Das, 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=RNAi+screening+reveals+requirement+for+host+cell+secretory+pathway+in+infection+by+diverse+families+of+negative-strand+RNA+viruses.">RNAi screening reveals requirement for host cell secretory pathway in infection by diverse families of negative-strand RNA viruses.</a> Proc. Natl. Acad. Sci. U. S. A. 108 (47), 19036-19041 (2011).</li><li>Lee, A. S. -. Y., Burdeinick-Kerr, R., Whelan, S. P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+small+interfering+RNA+screen+identifies+host+factors+required+for+vesicular+stomatitis+virus+infection.">A genome-wide small interfering RNA screen identifies host factors required for vesicular stomatitis virus infection.</a> J. Virol. 88 (15), 8355-8360 (2014).</li><li>Mahoney, D. J., Lefebvre, C., 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=Virus-tumor+interactome+screen+reveals+ER+stress+response+can+reprogram+resistant+cancers+for+oncolytic+virus-triggered+Caspase-2+cell+death.">Virus-tumor interactome screen reveals ER stress response can reprogram resistant cancers for oncolytic virus-triggered Caspase-2 cell death.</a> Cancer Cell. 20 (4), 443-456 (2011).</li><li>Mahoney, D. J., Stojdl, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+call+to+arms:+Using+RNAi+screening+to+improve+oncolytic+viral+therapy.">A call to arms: Using RNAi screening to improve oncolytic viral therapy.</a> Cytokine Growth Factor Rev. 21 (2-3), 161-167 (2010).</li><li>Mahoney, D. J., Stojdl, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomic+screening+to+enhance+oncolytic+virotherapy.">Functional genomic screening to enhance oncolytic virotherapy.</a> Br. J. Cancer. 108 (2), 245-249 (2012).</li><li>Allan, K. J., Stojdl, D. F., Swift, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+to+enhance+oncolytic+virus+immunotherapy.">High-throughput screening to enhance oncolytic virus immunotherapy.</a> Oncolytic Virother. 5, 15-25 (2016).</li><li>Lundholt, B. K., Scudder, K. M., Pagliaro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+reducing+edge+effect+in+cell-based+assays.">A simple technique for reducing edge effect in cell-based assays.</a> J. Biomol. Screen. 8 (5), 566-570 (2003).</li><li>Birmingham, A., Selfors, L. 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=Statistical+methods+for+analysis+of+high-throughput+RNA+interference+screens.">Statistical methods for analysis of high-throughput RNA interference screens.</a> Nat. Methods. 6 (8), 569-575 (2009).</li><li>Zhang, J., Chung, T., Oldenburg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+statistical+parameter+for+use+in+evaluation+and+validation+of+high+throughput+screening+assays.">A simple statistical parameter for use in evaluation and validation of high throughput screening assays.</a> J. Biomol. Screen. 4 (2), 67-73 (1999).</li><li>Huang, D. W., Sherman, B. T., Lempicki, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+and+integrative+analysis+of+large+gene+lists+using+DAVID+bioinformatics+resources.">Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.</a> Nat. Protoc. 4 (1), 44-57 (2009).</li><li>Huang, D. W., Sherman, B. T., Lempicki, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioinformatics+enrichment+tools:+paths+toward+the+comprehensive+functional+analysis+of+large+gene+lists.">Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists.</a> Nucleic Acids Res. 37 (1), 1-13 (2009).</li><li>Thomas, P. D., Campbell, M. J., 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=PANTHER:+a+library+of+protein+families+and+subfamilies+indexed+by+function.">PANTHER: a library of protein families and subfamilies indexed by function.</a> Genome Res. 13 (9), 2129-2141 (2003).</li><li>Szklarczyk, D., Morris, J. H., 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=The+STRING+database+in+2017:+quality-controlled+protein-protein+association+networks,+made+broadly+accessible.">The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible.</a> Nucleic Acids Res. 45 (D1), D362-D368 (2017).</li><li>Luc, P. -. V., Tempst, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINdb:+a+database+of+nuclear+protein+complexes+from+human+and+yeast.">PINdb: a database of nuclear protein complexes from human and yeast.</a> Bioinformatics. 20 (9), 1413-1415 (2004).</li><li>Chung, N., Zhang, X. D., 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=Median+absolute+deviation+to+improve+hit+selection+for+genome-scale+RNAi+screens.">Median absolute deviation to improve hit selection for genome-scale RNAi screens.</a> J. Biomol. Screen. 13 (2), 149-158 (2008).</li><li>Zhang, X. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illustration+of+SSMD,+z+Score,+SSMD*,+z*+Score,+and+t+statistic+for+hit+selection+in+RNAi+high-throughput+screens.">Illustration of SSMD, z Score, SSMD*, z* Score, and t statistic for hit selection in RNAi high-throughput screens.</a> J. Biomol. Screen. 16 (7), 775-785 (2011).</li><li>Zhang, X. D., Lacson, R., 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=The+use+of+SSMD-based+false+discovery+and+false+nondiscovery+rates+in+genome-scale+RNAi+screens.">The use of SSMD-based false discovery and false nondiscovery rates in genome-scale RNAi screens.</a> J. Biomol. Screen. 15 (9), 1123-1131 (2010).</li><li>Amberkar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+RNA+interference+screens+integrative+analysis:+towards+a+comprehensive+understanding+of+the+virus-host+interplay.">High-throughput RNA interference screens integrative analysis: towards a comprehensive understanding of the virus-host interplay.</a> World J. Virol. 2 (2), 18-31 (2013).</li><li>Barrows, N. J., Le Sommer, C., Garcia-Blanco, M. A., Pearson, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+reproducibility+between+genome-scale+siRNA-based+screens.">Factors affecting reproducibility between genome-scale siRNA-based screens.</a> J. Biomol. Screen. 15 (7), 735-747 (2010).</li><li>Carralot, J. -. P., Ogier, 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=A+novel+specific+edge+effect+correction+method+for+RNA+interference+screenings.">A novel specific edge effect correction method for RNA interference screenings.</a> Bioinformatics. 28 (2), 261-268 (2011).</li><li>Caffrey, D. R., Zhao, J., 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=siRNA+off-target+effects+can+be+reduced+at+concentrations+that+match+their+individual+potency.">siRNA off-target effects can be reduced at concentrations that match their individual potency.</a> PLoS ONE. 6 (7), e21503-e21511 (2011).</li><li>Makarenkov, V., Zentilli, P., Kevorkov, D., Gagarin, A., Malo, N., Nadon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+method+for+the+detection+and+elimination+of+systematic+error+in+high-throughput+screening.">An efficient method for the detection and elimination of systematic error in high-throughput screening.</a> Bioinformatics. 23 (13), 1648-1657 (2007).</li><li>Caraus, I., Alsuwailem, A. A., Nadon, R., Makarenkov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+and+overcoming+systematic+bias+in+high-throughput+screening+technologies:+a+comprehensive+review+of+practical+issues+and+methodological+solutions.">Detecting and overcoming systematic bias in high-throughput screening technologies: a comprehensive review of practical issues and methodological solutions.</a> Brief. Bioinform. 16 (6), 974-986 (2015).</li><li>Savidis, G., McDougall, W. 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=Identification+of+Zika+virus+and+dengue+virus+dependency+factors+using+functional+genomics.">Identification of Zika virus and dengue virus dependency factors using functional genomics.</a> Cell Rep. 16 (1), 232-246 (2016).</li><li>Ma, H., Dang, 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=A+CRISPR-Based+screen+identifies+genes+essential+for+West-Nile-virus-induced+cell+death.">A CRISPR-Based screen identifies genes essential for West-Nile-virus-induced cell death.</a> Cell Rep. 12 (4), 673-683 (2015).</li><li>Zhang, R., Miner, J. J., 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+CRISPR+screen+defines+a+signal+peptide+processing+pathway+required+by+flaviviruses.">A CRISPR screen defines a signal peptide processing pathway required by flaviviruses.</a> Nature. 535 (7610), 164-168 (2016).</li><li>Marceau, C. D., Puschnik, A. 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=Genetic+dissection+of+Flaviviridae+host+factors+through+genome-scale+CRISPR+screens.">Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens.</a> Nature. 535 (7610), 159-163 (2016).</li></ol>

在这里, 我们提出了一个使用高通量 rna 干扰筛选的协议, 以确定宿主目标, 可以操作, 以加强溶瘤病毒治疗。这已成功地通过溶瘤马拉巴病毒和溶瘤痘苗病毒进行了测试, 但是, 正如所指出的, 它可以适应其他溶瘤病毒或其他病毒的使用, 通常用于识别调节病毒复制的宿主基因。该筛选协议还旨在确定增加或减少传播的基因, 但它可以很容易地修改其他读数。例如, 我们的马拉巴病毒的屏幕被设计用来...

高通量全基因组 rna 检测已被证明是一个宝贵的工具, 以揭示生物洞察力在不同的研究领域, 包括病毒生物学。在过去的十年左右, 许多团体进行了基于细胞的大规模 rna 干扰筛查, 以广泛识别调节病毒复制的宿主基因 (在1,2,3,4,5,6, 7). 有趣的是, 大量的这些屏幕已经进行了癌症细胞和一些病毒, 目前溶瘤病毒候选包括痘苗病毒 8, 9, 10, 粘液瘤病毒11, 单纯疱疹病毒12, 水泡口炎病毒13,14和马拉巴病毒15。虽然大多数研究的重点是确定影响病毒复制的某些方面的宿主因素, 而不是确定增强溶瘤病毒治疗的 biotherapeutic 目标, 但有价值的数据可以从这些数据集中挖掘出来。这方面。很明显 , 确定可纵以加强溶瘤病毒治疗的宿主目标的强大潜力尚未得到充分利用。
目前, 大量的溶瘤病毒平台在临床前和临床研究中被测试, 其中包括单纯疱疹病毒、呼肠病毒和痘苗病毒, 这些都在后期试验中取得了明显的成功。对于大多数的这些平台, 已经致力于改变病毒基因组, 以加强治疗。例如, 为了增加肿瘤特异性, 特定的病毒基因已被删除或变异, 以显著限制病毒复制在正常细胞, 但不是在肿瘤细胞。在某些情况下, 转基因已被添加如 GM-脑脊液 (粒细胞巨噬素集落刺激因子), 以事实免疫应答或 NIS (碘化钠转运体), 使体内成像和细胞 radioiodide 吸收治疗目的.然而, 很少有工作专注于操纵宿主/肿瘤基因和探索宿主/肿瘤基因对溶瘤病毒复制的影响。随着高通量筛选技术的出现, 现在可以在基因组范围内探测宿主病毒相互作用, 并破译操作宿主基因组以增强溶瘤病毒治疗的机会 (16, 17,18)。
我们报告的第一项研究表明, 使用高通量基因筛选的概念证明, 以确定宿主目标, 可以操纵, 以加强溶瘤病毒治疗。为了发现调节马拉巴病毒裂癌的宿主基因, 该溶瘤 rhabdovirus 目前正在第一阶段和第二期试验中进行测试, 我们在三种不同的肿瘤细胞系中进行了全基因组 rna 检测, 并与 siRNA (短干扰 RNA) 重复。图书馆目标18120基因15。通过对筛查发现的通路进行分析, 揭示了 ER (内质网) 应力响应通路的富集。我们在这些路径中选择了10次命中, 以便使用 siRNA 与主屏幕中不同的目标序列进行二级验证。作为三级验证的一部分, 我们进行了 IRE1α (肌醇要求酶1α) 的抢救实验, 以确认命中目标。体外和体内使用 IRE1α的小分子抑制剂进行测试, 从而显著提高了溶瘤的功效。
Workenhe et 。12还使用一个聚集的慢病毒载体 shRNA (短发夹 RNA) 库对16056个基因进行了全基因组的 rna 干扰筛查, 以确定限制人疱疹病毒1型 (HSV-1) 突变体 KM100-mediated 裂癌的宿主因素癌细胞。在主屏幕上, 他们确定了343个基因的击倒, 导致 KM100-infected 细胞对模拟感染细胞的细胞毒性增强;他们选择了24这些基因进行二次验证。在24种基因中, 有8个基因在二次筛中被证实, 其中之一是 SRSF2 (丝氨酸/精氨酸-富含剪接因子 2), 随后在三级验证中使用基因抢救实验证实。该小组接着确定了一种 DNA 拓扑异构酶抑制剂化疗, 减少了 SRSF2 的磷酸化, 并表明 HSV-1 KM100 与此抑制剂的联合治疗导致了吐蕃肿瘤小鼠的延长生存。
在上述研究中, 遵循了类似的工作流程。每一个都是从一个主要的全基因组的 rna 干扰屏幕开始, 其次是在主屏幕上确定的点击次数中的辅助屏幕。其次是三级验证研究, 其中包括确认命中是在目标通过执行基因抢救实验在体外通过操纵目标基因产品在体内进行最终验证。在本文中, 我们首先提供了一个用于开发和验证高通量 siRNA 筛选试验的通用协议, 它涉及确定最佳转染条件、病毒数量和感染长度。我们还提供了一个详细的协议, 用于进行一个主要的高通量 siRNA 屏幕, 以及对执行次屏幕验证和三级验证的方法的总体描述。

<table>
	<tbody>
		<tr>
			<td>Consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>siRNA library</td>
			<td>GE Dharmacon</td>
			<td>G-005005-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 384-well plate</td>
			<td>Corning</td>
			<td>3985</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 384-well plate</td>
			<td>Becton Dickinson</td>
			<td>353962</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma</td>
			<td>W4502</td>
			<td></td>
		</tr>
		<tr>
			<td>siGenome SMARTpool PLK1</td>
			<td>GE Dharmacon</td>
			<td>M-003290-01</td>
			<td></td>
		</tr>
		<tr>
			<td>AllStars Negative Control siRNA</td>
			<td>Qiagen</td>
			<td>SI03650318</td>
			<td></td>
		</tr>
		<tr>
			<td>siGenome Non-targeting siRNA Pool #2</td>
			<td>GE Dharmacon</td>
			<td>D-001206-14-20</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/HIGH Glucose</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30022.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>F15051-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES solution (1M)</td>
			<td>Sigma</td>
			<td>H3535-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin 100X solution</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Thermo Fisher Scientific</td>
			<td>2500056</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS/Modified</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>13778100</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligofectamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>1225011</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>22600-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Resazurin sodium salt</td>
			<td>Sigma</td>
			<td>R7017-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Thermo Fisher Scientific</td>
			<td>H21492</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde (37% by weight)</td>
			<td>Fisher Scientific</td>
			<td>F79-4</td>
			<td></td>
		</tr>
		<tr>
			<td>500 ml bottles</td>
			<td>Corning</td>
			<td>430282</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive Sealing Film For Microplates</td>
			<td>Excel Scientific</td>
			<td>361006007</td>
			<td></td>
		</tr>
		<tr>
			<td>Peelable Heat Sealing Foil</td>
			<td>Thermo Fisher Scientific</td>
			<td>AB-3720</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTek MicroFlo Select Dispenser cassette</td>
			<td>Fisher Scientific</td>
			<td>11-120-625</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MicroFlo Select (liquid dispenser)</td>
			<td>Fisher Scientific</td>
			<td>11120621</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTek Synergy HT plate reader</td>
			<td>Biotek</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Opera Imaging and Analysis Instrument</td>
			<td>PerkinElmer</td>
			<td>HH10000115</td>
			<td></td>
		</tr>
		<tr>
			<td>KiNEDx Robotic Arm</td>
			<td>Peak Analysis and Automation (formerly Peak Robotics)</td>
			<td>KX-300-660</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytomat 24C Series Automated Incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>50080227</td>
			<td></td>
		</tr>
		<tr>
			<td>JANUS Automated Workstation</td>
			<td>PerkinElmer</td>
			<td>AJI4M01</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate Carousel</td>
			<td>Peak Analysis and Automation (formerly Peak Robotics)</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Alps 3000 plate sealer</td>
			<td>Thermo Fisher Scientific</td>
			<td>AB-3000</td>
			<td></td>
		</tr>
	</tbody>
</table>

genome-wide rnai screening to identify host factors that modulate oncolytic virus therapy

高通量全基因组 rna 干扰 (RNA 干涉) 筛选技术已被广泛用于发现影响病毒复制的宿主因素。在这里, 我们提出了这项技术的应用, 以揭露宿主目标, 专门调节复制的马拉巴病毒, 溶瘤 rhabdovirus, 和痘苗病毒的目的是加强治疗。虽然该协议已测试用于溶瘤马拉巴病毒和溶瘤痘苗病毒, 这种方法适用于其他溶瘤病毒, 也可以用来确定主机目标, 调节病毒复制的哺乳动物细胞在秘书长.本议定书介绍了哺乳动物细胞高通量 rna 干扰筛查的研究进展和验证, 主要的考虑因素和准备步骤对进行一次高通量的 rna 干扰屏幕, 以及一步一步指南为进行一次高通量的 rna 干扰屏幕;此外, 它概括地概述了进行二次屏幕验证和三级验证研究的方法。高通量 rna 干扰筛选的好处是, 它允许一个人以一种广泛而无偏见的方式对病毒复制的任何方面进行分类, 这些因素可以开发出一种体外检测方法, 如传染性、爆裂大小、和细胞毒性。它有能力揭露 biotherapeutic 的目标, 根据目前的知识。

在图 1中介绍了用于识别宿主目标以增强溶瘤病毒治疗的工作流的概述。
如图 1所示, 进行高通量干扰屏幕的关键第一步是检测开发。图 2A提供了用于转染优化分析的样板布局。图 2B由预期结果的代表性图像组成,图 2C</stron...

Watch this Scientific Journal Video about Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy at JoVE.com

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

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

在这里 , 我们描述了一种使用高通量 rna 干扰筛选的协议 , 以发现宿主目标可以作来增强溶瘤病毒治疗 , 特别是 rhabodvirus 和痘苗病毒治疗 , 但它可以很容易地适应其他溶瘤病毒平台或发现宿主基因, 通常调制病毒复制。

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

Research

JoVE Journal

Cancer Research

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

利用功能基因组筛选识别在肿瘤细胞球体培养潜在的新药物靶标

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

科研

癌症研究

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

流动 Cytometry-based 药物筛选系统对促进细胞分化胶质母细胞瘤小分子的鉴定

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

An Integrated Platform for Genome-wide Mapping of Chromatin States Using High-throughput ChIP-sequencing in Tumor Tissues

肿瘤组织中高通量芯片测序的染色质状态全基因组图谱集成平台

Histone modifications constitute a major component of the epigenome and play important regulatory roles in determining the transcriptional status of ...

Whole-body PET/MRI of Pediatric Patients: The Details That Matter

儿科患者全身 PET/MRI: 物质的细节

Integrated PET/MRI is a hybrid imaging technique enabling clinicians to acquire diagnostic images for tumor assessment and treatment monitoring with both ...

Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration

CRISPR 指南对小鼠前列腺病毒传递的基因改变

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) ...

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

一种检测慢性淋巴细胞白血病新 nfat2 靶基因的染色质免疫沉淀法

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

A Data Integration Workflow to Identify Drug Combinations Targeting Synthetic Lethal Interactions

数据集成工作流，以识别针对合成致命相互作用的药物组合

A synthetic lethal interaction between two genes is given when knock-out of either one of the two genes does not affect cell viability but knock-out of ...

Analyzing Tumor Gene Expression Factors with the CorExplorer Web Portal

分析肿瘤基因表达因子与CorExplorer门户网站

Differential gene expression analysis is an important technique for understanding disease states. The machine learning algorithm CorEx has shown utility ...

Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer

胃肠癌DNA甲基化基因组全谱分析

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

研究利普托梅宁格疾病直接靶向治疗的穆林·奥马亚异种格拉夫特模型

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...