Name: in vitro transcribed rna-based luciferase reporter assay to study translation regulation in poxvirus-infected cells
Uploaded: 2019-05-01T15:00:00
Description: Watch this Scientific Journal Video about In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells at JoVE.com

该项目由国家卫生研究院 (AI128406 www.nih.gov) 资助至 ZY, 部分由约翰逊癌症研究中心 (http://cancer.k-state.edu) 资助, 形式是研究生暑期津贴至 PD。

1. PCR 制备体外转录Dna 模板
<ol><li>要通过 PCR 制备 DNA 模板, 请设计引物。在设计引物时, 考虑引物长度、退火温度 (Tm)、gc 含量、3 ' 端 g 或 c 等关键特性。 请注意:在这些文献25、26、27 中详细讨论了这些文献。</li>
	<li>设计引物, 以生成 PCR 放大器包含以下元素在 5 ' 到 3 ' 的方向: T7-启动子, ?...

<ol>
	<li>Crick, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+protein+synthesis.">On protein synthesis.</a> Symposia of the Society for Experimental Biology. 12, 138-163 (1958).</li><li>Crick, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+Dogma+of+Molecular+Biology.">Central Dogma of Molecular Biology.</a> Nature. 227, 561-563 (1970).</li><li>Sonenberg, N., Hinnebusch, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Translation+Initiation+in+Eukaryotes:+Mechanisms+and+Biological+Targets.">Regulation of Translation Initiation in Eukaryotes: Mechanisms and Biological Targets.</a> Cell. 136, 731-745 (2009).</li><li>Spriggs, K. A., Bushell, M., Willis, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+Regulation+of+Gene+Expression+during+Conditions+of+Cell+Stress.">Translational Regulation of Gene Expression during Conditions of Cell Stress.</a> Molecular Cell. 40, 228-237 (2010).</li><li>Shchelkunov, S. N., Marennikova, S. S., Moyer, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Orthopoxviruses Pathogenic for Humans. , (2005).</li><li>Gale, M., Tan, S. L., Katze, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+Control+of+Viral+Gene+Expression+in+Eukaryotes.">Translational Control of Viral Gene Expression in Eukaryotes.</a> Microbiology and Molecular Biology Reviews. 64, 239-280 (2000).</li><li>Walsh, D., Mathews, M. B., Mohr, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tinkering+with+Translation:+Protein+Synthesis+in+Virus-Infected+Cells.">Tinkering with Translation: Protein Synthesis in Virus-Infected Cells.</a> Cold Spring Harbor Perspectives in Biology. 5, a012351 (2013).</li><li>Cao, S., Dhungel, P., Yang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Going+against+the+Tide:+Selective+Cellular+Protein+Synthesis+during+Virally+Induced+Host+Shutoff.">Going against the Tide: Selective Cellular Protein Synthesis during Virally Induced Host Shutoff.</a> Journal of Virology. 91, e00071-e00017 (2017).</li><li>Pelletier, J., Kaplan, G., Racaniello, V. R., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cap-independent+translation+of+poliovirus+mRNA+is+conferred+by+sequence+elements+within+the+5&#8217;+noncoding+region.">Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5&#8217; noncoding region.</a> Molecular and Cellular Biology. 8, 1103-1112 (1988).</li><li>Pelletier, J., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internal+initiation+of+translation+of+eukaryotic+mRNA+directed+by+a+sequence+derived+from+poliovirus+RNA.">Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA.</a> Nature. 334, 320-325 (1988).</li><li>Guo, L., Allen, E., Miller, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+a+cap-independent+translation+element+that+functions+in+either+the+3&#8242;+or+the+5&#8242;+untranslated+region.">Structure and function of a cap-independent translation element that functions in either the 3&#8242; or the 5&#8242; untranslated region.</a> RNA. 6, 1808-1820 (2000).</li><li>Simon, A. E., Miller, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3&#8242;+Cap-Independent+Translation+Enhancers+of+Plant+Viruses.">3&#8242; Cap-Independent Translation Enhancers of Plant Viruses.</a> Annual Review of Microbiology. 67, 21-42 (2013).</li><li>Marom, L., 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=Diverse+poly(A)+binding+proteins+mediate+internal+translational+initiation+by+a+plant+viral+IRES.">Diverse poly(A) binding proteins mediate internal translational initiation by a plant viral IRES.</a> RNA Biology. 6, 446-454 (2009).</li><li>Liu, B., Qian, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+reprogramming+in+cellular+stress+response.">Translational reprogramming in cellular stress response.</a> Wiley Interdisciplinary Review. RNA. 5, 301-305 (2014).</li><li>Ahn, B. Y., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capped+poly(A)+leaders+of+variable+lengths+at+the+5&#8217;+ends+of+vaccinia+virus+late+mRNAs.">Capped poly(A) leaders of variable lengths at the 5&#8217; ends of vaccinia virus late mRNAs.</a> Journal of Virology. 63, 226-232 (1989).</li><li>Ahn, B. Y., Jones, E. V., Moss, B. <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+the+vaccinia+virus+gene+encoding+an+18-kilodalton+subunit+of+RNA+polymerase+and+demonstration+of+a+5&#8217;+poly(A)+leader+on+its+early+transcript.">Identification of the vaccinia virus gene encoding an 18-kilodalton subunit of RNA polymerase and demonstration of a 5&#8217; poly(A) leader on its early transcript.</a> Journal of Virology. 64, 3019-3024 (1990).</li><li>Schwer, B., Visca, P., Vos, J. C., Stunnenberg, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discontinuous+transcription+or+RNA+processing+of+vaccinia+virus+late+messengers+results+in+a+5&#8242;+poly(A)+leader.">Discontinuous transcription or RNA processing of vaccinia virus late messengers results in a 5&#8242; poly(A) leader.</a> Cell. 50, 163-169 (1987).</li><li>Yang, Z., Martens, C. A., Bruno, D. P., Porcella, S. F., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pervasive+initiation+and+3&#8242;+end+formation+of+poxvirus+post-replicative+RNAs.">Pervasive initiation and 3&#8242; end formation of poxvirus post-replicative RNAs.</a> Journal of Biological Chemistry. 287, 31050-31060 (2012).</li><li>Dhungel, P., Cao, S., Yang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+5&#8217;-poly(A)+leader+of+poxvirus+mRNA+confers+a+translational+advantage+that+can+be+achieved+in+cells+with+impaired+cap-dependent+translation.">The 5&#8217;-poly(A) leader of poxvirus mRNA confers a translational advantage that can be achieved in cells with impaired cap-dependent translation.</a> PLOS Pathogens. 13, e1006602 (2017).</li><li>Jha, 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=Trans-kingdom+mimicry+underlies+ribosome+customization+by+a+poxvirus+kinase.">Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase.</a> Nature. 546, 651-655 (2017).</li><li>Dai, 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=Ribosome+Profiling+Reveals+Translational+Upregulation+of+Cellular+Oxidative+Phosphorylation+mRNAs+during+Vaccinia+Virus-Induced+Host+Shutoff.">Ribosome Profiling Reveals Translational Upregulation of Cellular Oxidative Phosphorylation mRNAs during Vaccinia Virus-Induced Host Shutoff.</a> Journal of Virology. 91, e01858-e01816 (2017).</li><li>De Silva, F. S., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin-independent+plasmid+replication+occurs+in+vaccinia+virus+cytoplasmic+factories+and+requires+all+five+known+poxvirus+replication+factors.">Origin-independent plasmid replication occurs in vaccinia virus cytoplasmic factories and requires all five known poxvirus replication factors.</a> Journal of Virology. 2, 23 (2005).</li><li>Yang, Z., Bruno, D. P., Martens, C. A., Porcella, S. F., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+high-resolution+analysis+of+vaccinia+virus+and+host+cell+transcriptomes+by+deep+RNA+sequencing.">Simultaneous high-resolution analysis of vaccinia virus and host cell transcriptomes by deep RNA sequencing.</a> Proceedings of the National Academy of Sciences. 107, 11513-11518 (2010).</li><li>Yang, Z., Bruno, D. P., Martens, C. A., Porcella, S. F., Moss, B. <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+Analysis+of+the+5&#8242;+and+3&#8242;+Ends+of+Vaccinia+Virus+Early+mRNAs+Delineates+Regulatory+Sequences+of+Annotated+and+Anomalous+Transcripts.">Genome-Wide Analysis of the 5&#8242; and 3&#8242; Ends of Vaccinia Virus Early mRNAs Delineates Regulatory Sequences of Annotated and Anomalous Transcripts.</a> Journal of Virology. 85, 5897-5909 (2011).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+Chain+Reaction:+Basic+Protocol+Plus+Troubleshooting+and+Optimization+Strategies.">Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies.</a> Journal of Visualized Experiments. , (2012).</li><li>Dieffenbach, C. W., Lowe, T. M., Dveksler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+concepts+for+PCR+primer+design.">General concepts for PCR primer design.</a> PCR Methods and Applications. 3, S30-S37 (1993).</li><li>Innis, M. A., Gelfand, D. H., Sninsky, J. J., White, T. 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> PCR Protocols: A Guide to Methods and Applications. , (2012).</li><li>Baklanov, M. M., Golikova, L. N., Malygin, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+on+DNA+Transcription+of+Nucleotide+Sequences+Upstream+to+T7+Promoter.">Effect on DNA Transcription of Nucleotide Sequences Upstream to T7 Promoter.</a> Nucleic Acids Research. 24, 3659-3660 (1996).</li><li>Thornton, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+by+Overlap+Extension+PCR+to+Obtain+Hybrid+DNA+Products.">Splicing by Overlap Extension PCR to Obtain Hybrid DNA Products.</a> The Genetic Manipulation of Staphylococci: Methods and Protocols. , 43-49 (2017).</li><li>Akichika, 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=Cap-specific+terminal+N6-methylation+of+RNA+by+an+RNA+polymerase+II&#8211;associated+methyltransferase.">Cap-specific terminal N6-methylation of RNA by an RNA polymerase II&#8211;associated methyltransferase.</a> Science. , (2018).</li><li>Sun, H., Zhang, M., Li, K., Bai, D., Yi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cap-specific,+terminal+N6-methylation+by+a+mammalian+m6Am+methyltransferase.">Cap-specific, terminal N6-methylation by a mammalian m6Am methyltransferase.</a> Cell Research. 1, (2018).</li><li>Boulias, K., 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+the+m6Am+methyltransferase+PCIF1+reveals+the+location+and+functions+of+m6Am+in+the+transcriptome.">Identification of the m6Am methyltransferase PCIF1 reveals the location and functions of m6Am in the transcriptome.</a> bioRxiv. , 485862 (2018).</li><li>Dominissini, D., Rechavi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N4-acetylation+of+Cytidine+in+mRNA+by+NAT10+Regulates+Stability+and+Translation.">N4-acetylation of Cytidine in mRNA by NAT10 Regulates Stability and Translation.</a> Cell. 175, 1725-1727 (2018).</li><li>Wei, 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=Differential+m6A,+m6Am,+and+m1A+Demethylation+Mediated+by+FTO+in+the+Cell+Nucleus+and+Cytoplasm.">Differential m6A, m6Am, and m1A Demethylation Mediated by FTO in the Cell Nucleus and Cytoplasm.</a> Molecular Cell. 71, 973-985 (2018).</li><li>Fu, Y., Dominissini, D., Rechavi, G., He, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+regulation+mediated+through+reversible+m6A+RNA+methylation.">Gene expression regulation mediated through reversible m6A RNA methylation.</a> Nature Reviews Genetics. 15, 293-306 (2014).</li></ol>

所有四核步骤都是体外转录 rna 基荧光素酶报告法成功的关键。应特别注意引物设计, 特别是 T7 启动子序列。T7 RNA 聚合酶开始转录从在 5 '-utr 序列之前添加的 T7 启动子中的下划线第一 g (gGg-5 '-utr-aug-)。虽然转录起始位点 (TSS) 从 5 ' 端的第一个 G 开始, 但在 T7 启动子区域中, 减少 G 的小于3的数量会减少体外转录的 RNA 产量/输出。在实验中, 我们观察到凝胶纯化 DNA 产物不是最佳的体外转录, 因?...

根据中心教条, 遗传信息从 dna 流向 rna, 然后最后流向蛋白质1,2。遗传信息的这种流动在许多层面上受到高度调控, 包括 mrna 翻译3,4。研究用记者检测来衡量基因表达的调控, 将有助于了解这一过程中涉及的调控机制。在这里, 我们描述了一种研究 mrna 翻译的方案, 使用体外转录 rna 为基础的荧光素酶报告法在 pxvirs 感染细胞。
痘病毒包括许多高度危险的人类和动物病原体5。与所有其他病毒一样, 弓形病毒完全依靠宿主细胞机制进行蛋白质合成6,7,8。为了有效地合成病毒蛋白, 病毒进化出许多策略来劫持细胞翻译机制, 将其重定向, 用于病毒 rna 7,8的翻译。病毒常用的一种机制是在其转录中使用cis作用元素。值得注意的例子包括内部核糖体进入网站 (ires) 和与上限无关的翻译增强剂 (cite)9、10、11。这些cis元素通过通过不同的机制吸引翻译机械 12,13, 14,使病毒转录具有平移优势。超过 100个 poxvirus mrna 在 5 '-未翻译区域 (5 '-utr) 中具有进化保守的cis作用元素: 在这些 mrna15,16的 5 '-端的 5 '-多边形 (a) 引线。这些 5 ' poly (a) 引线的长度是异质性的, 是由 poxvirus-c金编码 rna 聚合酶在转录17,18 期间的滑移产生的。我们和其他人最近发现, 5 ' poly (a) 领导者赋予 mrna 在感染疫苗病毒 (vacv) 的细胞的翻译优势, 该病毒是天花病毒 19,20的典型成员。
体外转录 rna 基荧光素酶报告法初步开发, 以了解 5 '-聚 (a) 引线在猪病毒感染19,21期间 mrna 翻译中的作用。尽管基于粒细胞 dna 的荧光素酶记者检测已被广泛使用, 但有几个缺点会使感染 poxvirus-infected 细胞的结果解释复杂化。首先, 质粒能够在受 vac 感染的22细胞中复制。其次, 神秘转录往往发生在质粒dna 18,23,24。第三, VAC 启动子驱动转录产生聚 (A)-异质长度的引线, 因此在一些实验18中难以控制聚 (A) 引线长度。体外转录 rna 酶报告法规避这些问题, 数据解释很简单。
该方法有四个关键步骤: (1) 聚合酶链反应 (PCR) 生成体外转录的 DNA 模板;(2) 体外转录生成 mRNA;(3) 转染, 将 mRNA 输送到细胞中;(4) 检测荧光素酶活性作为翻译指标 (图 1)。生成的 PCR 放大器包含 5 ' 到 3 ' 方向中的以下元素: T7-tokor、poly (A) 引线或所需的 5 '-utr 序列、萤火虫荧光酶开放阅读框架 (ORF), 然后是聚 (A) 尾。以 PCR 放大器为模板, 利用 T7 聚合酶进行体外转录合成 mRNA。在体外转录过程中,mRNAg 帽或其他盖帽模拟被纳入新合成的 mrna 中。封顶的转录被转移到未经感染或没有感染的 vacv 细胞。细胞裂解液在转染后的所需时间被收集, 以测量表明转染 mRNA 产生蛋白质的荧光素酶活性。本报告法可用于研究 5 '-utr、3 '-utr 或其他区域 mRNA 中的cis元素的翻译调控。此外, 基于体外转录 rna 的检测方法可用于研究不同的翻译起始机制, 包括与上限相关的起始、与盖帽无关的起始、重新启动和像 RES 这样的内部起始。

<table>
	<tbody>
		<tr>
			<td>2X-Q5 Master mix</td>
			<td>New England Biolabs</td>
			<td>M0492</td>
			<td>High-Fidelity DNA Polymerase used in PCR</td>
		</tr>
		<tr>
			<td>3&#180;-O-Me-m7G(5&#39;)ppp(5&#39;)G RNA Cap Structure Analog</td>
			<td>New England Biolabs</td>
			<td>S1411L</td>
			<td>Anti reverse Cap analog or ARCA</td>
		</tr>
		<tr>
			<td>Corning 96 Well Half-Area white flat bottom polystyrene microplate</td>
			<td>Corning</td>
			<td>3693</td>
			<td>Opaque walled 96 well white plate with solid bottom</td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega</td>
			<td>E1960</td>
			<td>Dual-Luciferase Assay Kit (DLAK)</td>
		</tr>
		<tr>
			<td>E.Z.N.A. Cycle Pure Kit</td>
			<td>OMEGA BIO-TEK</td>
			<td>D6492</td>
			<td>PCR purification kit</td>
		</tr>
		<tr>
			<td>GloMax Navigator Microplate Luminometer</td>
			<td>Promega</td>
			<td>GM2010</td>
			<td>Referred as multimode plate reader luminometer</td>
		</tr>
		<tr>
			<td>HiScribe T7 Quick High Yield RNA synthesis Kit</td>
			<td>New England Biolabs</td>
			<td>E2050S</td>
			<td>In-Vitro transcription kit</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Thermo Fisher Scientific</td>
			<td>11668019</td>
			<td>Cationic lipid transfection reagent</td>
		</tr>
		<tr>
			<td>NanoDrop2000</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-2000</td>
			<td>Used to measure DNA and RNA concentration</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985070</td>
			<td>Reduced serum media</td>
		</tr>
		<tr>
			<td>Purelink RNA Mini Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>12183018A</td>
			<td>RNA purification kit</td>
		</tr>
		<tr>
			<td>Vaccinia Capping System</td>
			<td>New England Biolabs</td>
			<td>M2080</td>
			<td>Capping system</td>
		</tr>
	</tbody>
</table>

in vitro transcribed rna-based luciferase reporter assay to study translation regulation in poxvirus-infected cells

病毒 DNA 复制后转录的每一个痘病毒 mRNA 都有一个进化保守的、非模板化的 5 '-聚 (A) 引线在 5 '-utr 中。为了剖析 5 '-聚 (A) 引线在天花病毒感染过程中 mRNA 翻译中的作用, 我们开发了一种体外转录 rna 基荧光素酶报告法。本报告分析包括四个核心步骤: (1) PCR 扩增 DNA 模板进行体外转录;(2) 利用 T7 RNA 聚合酶进行体外转录生成 mRNA;(3) 转染, 将体外转录的 mRNA 引入细胞;(4) 检测荧光素酶活性作为翻译指标。本文介绍的基于 rna 的荧光素酶报告法规避了弓形虫感染细胞中质粒复制和质粒中的神秘转录问题。该协议可用于确定 mRNA 中的 cis元素在 poxvirus-infected 感染细胞以外的系统中的翻译调控, 包括 5 '-utr 和 3 '-utr。此外, 还可以利用这种方法研究不同的翻译启动模式, 如与上限相关、与上限无关、重新启动和内部启动。

体外转录 rna 基荧光素酶报告法的四个步骤: PCR 生成 DNA 模板体外转录, 体外转录生成 mRNA, mRNA 转染, 和荧光素酶测量, 可以在示意图中看到 (图 1)。图中说明了 DNA 模板 (Fluc 和 Rluc) 引物的设计和悬垂扩展 PCR 的一般方案 (图 2a)。PCR 后, TAE 琼脂糖凝胶电泳检测出正确大小的 PCR 产物 (图 2B)。随后, PCR 产物被用?...

Watch this Scientific Journal Video about In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells at JoVE.com

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

我们提出了一种研究 mrna 翻译调节的方案, 在天花病毒感染细胞使用体外转录 Rna 基荧光素酶报告法。该方法可用于研究 mRNA 的cis元素的翻译调控, 包括 5 '-未翻译区域 (utr) 和 3 '-utr。使用此方法还可以检查不同的翻译启动模式。

体外转录 rna 基荧光素酶记者法研究类病毒感染细胞的翻译规律

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To ...

我们关于体外转录RNA的荧光素酶测定的协议有助于研究痘病毒感染细胞的转化调控。此外，Poly（A）升的定制长度允许研究其对翻译的监管影响。该技术的主要优点是，它允许研究Cis元素的翻译调控，如5'UTR和3'UTR在mRNA和广泛的翻译启动。体外转录RNA基荧光素酶检测方法可进行定制，以纳入对盖和其他修饰的修改，并用于测试对翻译的影响。首先，设计前进和反向底转，以生成包含以下元素的 PCR 放大器，方向为 5'到 3'：T7-促进器， 聚（A）升，萤火虫路西加酶 ORF 在聚（A）尾称为T7_12A-Fluc，包括几个额外的核苷酸在前进底转，其次是T7-促进器聚（A）升或所需的5'UTR序列和大约20核苷酸对应于5'端的记者基因的OF。根据与报告基因的 ORF 的 5'端对应的熔化温度调整底因长度，确保底源器中的相应区域与基因的感应链相同。然后设计反向底转，包括聚（A）尾和大约20个核苷酸。根据与报告基因的 ORF 的 3'end 对应的熔化温度进行调整，确保底原体的相应区域与基因的反感链相同，且在 Poly（A） 尾部之前存在帧内停止 codon。对于内部控制，设计另一组底像，包含以下元素在5'到3'方向：T7-启动器，一个随机的5'UTR编码序列包含科扎克序列，雷尼拉路西法酶 ORF和聚（A）尾，称为T7_Kozak-Rluc。要设置反应，请按以下顺序将试剂添加到 PCR 管中：无脱水、2X 高保真DNA聚合酶、底注和序列确认的荧光素酶模板 DNA。经过标准的三步PCR循环生成DNA模板后，通过运行5-10%的PCR反应，在1%的agarose TAE凝胶电泳和分子量标准中检测PCR产品。要确定 PCR 产品的大小，请将凝胶可视化为紫外线照明器下。在确定 PCR 产品的正确尺寸后，使用市售的 PCR 纯化套件使用 100 微升无核酸酶无素水进行纯化，以检测 DNA。使用分光光度计检查纯化DNA的浓度，并将其储存在零下20摄氏度，立即用于体外转录。要从PCR产品合成RNA，请将以下试剂加入微离心管：DNase-RNase无水、NTP缓冲液混合、帽模拟、PCR产品模板和体外转录试剂盒中的T7-RNA聚合酶混合。彻底混合。在37摄氏度下孵育两小时，然后使用RNA纯化试剂盒进行合成RNA的纯化。要检查纯化的RNA，在70度加热RNA5分钟，去除二次结构，并在1.5阿加罗斯TBE凝胶上运行。然后在紫外线照明器下可视化凝胶。使用分光光度计检查纯化RNA的浓度。阿里语，储存在零下80摄氏度。在24个井板中播种 HeLa 细胞，第二天达到 80-90% 的汇合，并在 37 摄氏度的培养箱中孵育过夜，二氧化碳含量为 5%。感染HLA细胞与瓦奇尼亚病毒在五个感染的多重和保持未感染的希拉细胞进行比较。然后在37摄氏度下用5%的二氧化碳孵育，10至12小时。感染后所需小时后，将12A序列的480纳米图与携带萤火虫路西法酶mRNA的180纳米图和携带蕾妮拉路西法酶mRNA的20纳米图混合在一个微离心管中。在另一个微离心管中，加入1.1微升的阳离子脂质转染试剂。将 55 微升的减少血清介质添加到两个管中，在室温下混合和孵育五分钟。然后加入55微升阳离子脂质转染试剂含有减少血清介质到mRNA包含管。与移液器轻轻但彻底混合，在室温下孵育15分钟。在孵育过程中，从细胞中去除细胞培养培养，每井加入400微升的血清培养。孵育完成后，在24个井板的井中，每井均匀地加入100微升混合物滴。与12A Fluc和Kozak Rluc mRNA共同感染后5小时，从细胞中去除还原的血清介质，通过从Luciferase测定试剂盒中加入150微升1X解液缓冲液来细胞酶化。在室温下孵育10分钟后，使用橡胶和无菌注射器刮取细胞，并转移到微离心管。为了颗粒细胞碎片，在摄氏4度下将12，000次的落酸离心10分钟。每井输送30微升清量，用于具有坚固底部的不透明壁壁96井白色测定板。使用路西法酶测定套件和多模板读卡器发光计使用手稿中描述的动力学功能测量双发光。将发光读取数据导出为理想的文件格式后，通过将 Fluc 值除以内部控制 Rluc 值，确定未感染和 VACV 感染 HeLA 细胞中的 12A Fluc mRNA 的相对平移率。此检测用于测试在未感染和 VACV 感染的细胞中含有 5'Poly（A） 升的 Fluc mRNA 的翻译效率。未感染细胞和VACV感染细胞均成功与氟克和Rluc mRNA共同感染。将氟化物除以Rluc使细胞RNA稳定性的转染效率正常化。与未感染的细胞相比，5'Poly（A）升含有mRNA在VACV感染期间具有转化优势。这不是由于差异转染效率或mRNA稳定性，因为RNA水平在未感染和VACV感染细胞中相似，在mRNA转染后5小时。在设计底像时要特别小心，因为这不仅会影响 PCR 反应效率，还会影响所有需要扩增 DNA 的下游过程。该方法是快速测试Cis元素翻译规制的重要检测方法。如果可能，此方法应得到其他补充实验的证实。使用瓦奇尼亚病毒时应采取预防措施，避免接触溴化乙基和苯酚等化学物质。

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Immunology and Infection

Unit 1

Translation: RNA to Protein

SCIENTISTS IN ACTION

Using Luciferase to Image Bacterial Infections in Mice

用荧光素酶在小鼠细菌性感染的形象

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

科研

免疫与感染

Seven Steps to Stellate Cells

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

到哺乳动物的上皮细胞在体外细菌粘附的含量

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

一个<em&gt;在体外</em&gt;共感染模型研究<em&gt;恶性疟原虫</em人原发性单核细胞源性的免疫细胞-HIV-1相互作用

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

在体外和体内模型来研究细菌粘附于血管壁下流动条件

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

一种新的体外创面愈合试验评价细胞迁移

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...