Name: retroviral scanning: mapping mlv integration sites to define cell-specific regulatory regions
Uploaded: 2017-05-28T14:00:00.000+00:00
Duration: 10 min 10 s
Description: Watch this Scientific Journal Video about Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions at JoVE.com

这项工作由欧洲研究委员会（ERC-2010-AdG，GT-SKIN），意大利教育，大学和研究部（2010年Ricerca的FIRB-Futuro-RBFR10OS4G，2012年Ricerca的FIRB-Futuro-RBFR126B8I_003）的赠款支持，EPIGEN Epigenomics旗舰项目），意大利卫生部（Young Research Call 2011 GR-2011-02352026）和Imagine研究所基金会（法国巴黎）。

人类细胞的MLV转导<ol><li> 分离靶细胞并用携带eGFP报告基因的mLV衍生的逆转录病毒载体转导它们，并用水泡性口炎病毒G（VSV-G）或单调性包膜糖蛋白16假型。 <ol><li>将模拟转导细胞作为阴性对照进行以下分析。由于基于mLV的逆转录病毒载体可以转导有效分裂的细胞，在刺激细胞分裂的条件下培养靶细胞群体。需要针对研究中的每种细胞类型，特异性优化转导条件。人造血祖细胞，T细胞和表皮细胞的细胞生长和转导条件描述于参考文献7,14,15和17中。 </li></ol></li><li>转导后48 h，在300μL含有2％胎牛血清的磷酸盐缓冲盐水（PBS）中重悬10 000个细胞，并用f低细胞分析（488nm激发激光）。使用模拟转导的细胞作为阴性对照。为了优化整合位点检索，通过荧光活化细胞分选（FACS）纯化GFP +细胞。 </li><li>收集0.5-5百万个细胞用于基因组DNA制备。在分析真正干细胞的长期后代时，较长的培养期（&gt; 7天）优先稀释未整合的原病毒并且是必需的（参见讨论部分和参考文献15 ）。细胞颗粒可以快速冷冻并储存在-80°C直到使用。 </li></ol> 2.通过接头介导的PCR（LM-PCR）扩增mLV整合位点<ol><li> 基因组DNA（gDNA）的制备 <ol><li>使用基于柱的DNA提取试剂盒提取gDNA，并按照制造商的说明遵循培养细胞的方案。 </li></ol></li><li> 限制酶消化上<ol><li>在1.5 mL管中每个样品设置4个限制酶消化。通过加入1μLTru9I（10U）和1μL缓冲液M，最终体积为10μL，每个管中消化0.1至1μggDNA。将反应在65℃下孵育6小时或过夜。 </li><li>向每个反应物中加入1μlPstI（10U），1μL缓冲液H和8μL水。将反应在37℃孵育6小时或过夜。样品可以保存在-20°C直到使用。 </li></ol></li><li> 连接器结扎 <ol><li>通过以100μM的浓度混合连接体加链和接头负链寡核苷酸，在1.5mL管中制备100μMTru9I连接体储备溶液。将管置于100°C的水中，并在室温下冷却。 Tru9I接头储液可以在-20°C储存，直到使用。 </li><li>在1.5 mL管中设置8个接头连接反应。对于每个反应，添加以下组分s至10μL限制酶消化:1.4μL10X T4 DNA连接酶反应缓冲液，1μL10μM接头Tru9I，1μL（2,000U）T4DNA连接酶和0.6μL水。在16℃下孵育3至6小时。样品可以保存在-20°C直到使用。 </li></ol></li><li> 第一次PCR <ol><li>在0.2mL管中设置48个PCR反应（来自每个连接管的6个反应）。对于每个PCR，将以下试剂加入2μL连接反应:5μL10X PCR缓冲液，2μL50mM硫酸镁，1μL10mM脱氧核苷酸（dNTP）Mix，1μL10μM接头引物1 μL10μMmLV-3&#39;LTR引物，0.3μL（1.5U）Taq DNA聚合酶和37.7μL水。引物序列在表1中提供。 </li><li>在具有加热盖的热循环仪中进行PCR反应，如下:95℃2分钟; 25个循环，95℃15秒，55℃30秒，72℃1分钟; 72°; C为5分钟;保持在4°C。样品可以保存在-20°C直到使用。 </li></ol></li><li> 第二次PCR <ol><li>在0.2 mL管中设置48个PCR反应（每个"第一PCR"管1个）。对于每个PCR，将以下组分添加到2μL第一次PCR反应中:5μL10X PCR缓冲液，2μL50mM硫酸镁，1μL10mM dNTP Mix，1μL10μM接头嵌套引物，1 μL10μMmLV-3&#39;LTR嵌套引物，0.3μLTaq DNA聚合酶（1.5U）和37.7μL水。使用针对特定大规模测序策略设计的嵌套引物:（i）与Illumina平台兼容的接头嵌套引物和mLV-3&#39;LTR嵌套引物; （ii）接头嵌套引物和与Roche平台兼容的mLV-3&#39;LTR嵌套引物。引物序列列于表1 。 </li><li>在具有加热盖的热循环仪中进行PCR反应，如下:95℃2分钟; 25循环95℃15秒，58℃30秒，72℃1分钟; 72℃5分钟;保持在4°C。样品可以保存在-20°C直到使用。 </li><li>将48个巢式PCR反应置于15 mL试管（最终体积〜2.4 mL）中。样品可以保存在-20°C直到使用。 </li></ol></li><li> 通过琼脂糖凝胶电泳确定LM-PCR产物的存在和大小。 <ol><li>加入4μL载体缓冲液至20μLLM-PCR产物，并将样品加载到1％琼脂糖凝胶上，并连同100 bp DNA梯。以5 V / cm的速度运行凝胶30至60分钟，并通过溴化乙锭染色观察PCR产物。 </li><li>从模拟转导的样品中进行LM-PCR反应作为阴性对照。 </li></ol></li><li> 通过加入0.1体积的乙酸钠溶液（3M; pH5.2）和2.5体积的100％乙醇沉淀扩增子。在-80℃下混合并冷冻20分钟。 <ol><li>旋转在福在4℃下在标准微量离心机中速度20分钟。弃去上清液，用70％乙醇洗涤沉淀。在标准微量离心机中以4℃全速旋转5分钟。 </li><li>弃上清，空气干燥沉淀。加入200μLPCR级水，并重悬DNA。样品可以保存在-20°C直到使用。 </li></ol></li><li> 向100μLLM-PCR产物中加入20μL载体缓冲液，并将样品加载到1％琼脂糖凝胶上，并连同100 bp DNA梯。 <ol><li>以5V / cm的速度运行凝胶30至60分钟，并切割含有150至500bp长的扩增子的部分凝胶。 </li><li>使用柱式凝胶提取试剂盒纯化LM-PCR产物，并使用紫外分光光度计测定浓度。 </li></ol></li><li>根据制造商的说明，使用1μL的文库来评估使用生物分析仪器的LM-PCR产品的长度。 </li><li><strong&gt;霰弹枪克隆扩增子<ol><li>使用20 ng纯化的LM-PCR产物，并按照制造商的说明将其克隆到pCR2.1-TOPO载体中。 </li><li>使用M13通用引物进行测序反应，随后对所得序列进行基因组测绘，以显示&gt; 50％克隆中病毒 - 基因组连接（包括mLV-3&#39;LTR嵌套引物）的存在，以鉴定合适的样品用于大规模测序。病毒 - 基因组连接的实例: <img alt="连接点" src="/files/ftp_upload/55919/55919junction.jpg" /> MLV-3&#39;LTR嵌套引物454和接头嵌套引物454（ 表1 ）以粗体表示，病毒LTR的3&#39;末端以斜体表示。人类基因组序列以红色文本（chr10:6439408-6439509，hg19）突出显示。 </li></ol></li></ol> mLV集成位点的大规模测序注意:LM-PCR产品可以使用商业平台（在第二次PCR反应中选择适当的嵌套引物对，参见小节2.5.1）进行测序。对于罗氏GS-FLX焦磷酸测序平台的测序，请参考以前的论文7,14,15。在本节中，描述了用于Illumina测序平台的新优化协议。 <ol><li> 图书馆准备 <ol><li>每个样品在0.2 mL管中设置1个分选PCR反应。对于每个PCR，向5μL（150-170 ng）纯化的LM-PCR产物中加入以下试剂:5μLIndex Primer 1，5μLIndex Primer 2，25μL2X Master Mix和10μLPCR-等级水。对每个样本使用不同的索引组合。 </li><li>在具有加热盖的热循环仪中进行PCR反应，如下:95℃3分钟; 95℃30秒，55℃30秒，72℃30秒的8个循环; 72°C 5 m在;保持在4°C。 </li><li>使用固相可逆固定（SPRI）珠分离方法纯化PCR产物:在新的1.5 mL管中，向每个样品加入56μL珠，并按照制造商的说明进行操作。在25μLTris-HCl 10mM中洗脱。库可以保存在-20°C直到使用。 </li></ol></li><li> 图书馆检查 <ol><li>使用1μL样品，使用生物分析仪器来评估文库大小。 </li><li>根据制造商的说明，使用1μL样品，使用基于荧光的实时PCR检测来定量文库的摩尔浓度。 </li></ol></li><li> 图书馆稀释和测序 <ol><li>使用Tris-HCl 10mM将文库稀释至10nM。对于汇集文库，将5μL每个稀释的文库转移到新的1.5mL管中，然后在10mM Tris-HCl中将池稀释至4nM。 </li><li>将5μL稀释池与5μL0.2 N NaOH混合，加入1.5 mL管，短暂涡旋，旋转并在室温下孵育5分钟以使文库变性。 </li><li>将管放在冰上，加入990μL预冷的杂交缓冲液（HT1）。将等分试样在新的1.5 mL管中加入300μL变性池，加入300μL预冷HT1，获得10 pM的最终文库池。 </li><li>同时，在1.5 mL管中混合2μLPhiX对照文库（10 nM）和3μLTris-HCl 10 mM。加入5μLNaOH 0.2 N，短暂旋转，旋转下来，室温孵育5 min，使稀释的PhiX变性。将管放在冰上，加入990μL预冷HT1。 </li><li>在新的1.5 mL管中分装300μL变性PhiX，并加入300μL预冷HT1，以获得10 pM的最终PhiX。 </li><li>在新的1.5 mL管中，将510μL变性库文库与90μL变性PhiX文库混合，从而获得最终10 pM池，其中15％为PhiX对照。 </li><li>移取600μL样品体积进入解冻的测序试剂盒的Load Sample储存器，并立即执行单次读取150循环运行。 注意:本协议所需的关键试剂和引物序列见表1 。 </li></ol></li></ol>

<ol>
	<li>Ernst, 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=Mapping+and+analysis+of+chromatin+state+dynamics+in+nine+human+cell+types.">Mapping and analysis of chromatin state dynamics in nine human cell types.</a> Nature. 473 (7345), 43-49 (2011).</li><li>Shlyueva, D., Stampfel, G., Stark, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+enhancers:+from+properties+to+genome-wide+predictions.">Transcriptional enhancers: from properties to genome-wide predictions.</a> Nat Rev Genet. 15 (4), 272-286 (2014).</li><li>Roadmap Epigenomics Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+analysis+of+111+reference+human+epigenomes.">Integrative analysis of 111 reference human epigenomes.</a> Nature. 518 (7539), 317-330 (2015).</li><li>Heintzman, N. 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=Histone+modifications+at+human+enhancers+reflect+global+cell-type-specific+gene+expression.">Histone modifications at human enhancers reflect global cell-type-specific gene expression.</a> Nature. 459 (7243), 108-112 (2009).</li><li>Creyghton, M. P., 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=Histone+H3K27ac+separates+active+from+poised+enhancers+and+predicts+developmental+state.">Histone H3K27ac separates active from poised enhancers and predicts developmental state.</a> Proc Natl Acad Sci U S A. 107 (50), 21931-21936 (2010).</li><li>Hnisz, 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=Super-enhancers+in+the+control+of+cell+identity+and+disease.">Super-enhancers in the control of cell identity and disease.</a> Cell. 155 (4), 934-947 (2013).</li><li>Cattoglio, 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=High-definition+mapping+of+retroviral+integration+sites+identifies+active+regulatory+elements+in+human+multipotent+hematopoietic+progenitors.">High-definition mapping of retroviral integration sites identifies active regulatory elements in human multipotent hematopoietic progenitors.</a> Blood. 116 (25), 5507-5517 (2010).</li><li>Biasco, 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=Integration+profile+of+retroviral+vector+in+gene+therapy+treated+patients+is+cell-specific+according+to+gene+expression+and+chromatin+conformation+of+target+cell.">Integration profile of retroviral vector in gene therapy treated patients is cell-specific according to gene expression and chromatin conformation of target cell.</a> EMBO Mol Med. 3 (2), 89-101 (2011).</li><li>De Ravin, S. 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=Enhancers+are+major+targets+for+murine+leukemia+virus+vector+integration.">Enhancers are major targets for murine leukemia virus vector integration.</a> J Virol. 88 (8), 4504-4513 (2014).</li><li>LaFave, M. 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=mLV+integration+site+selection+is+driven+by+strong+enhancers+and+active+promoters.">mLV integration site selection is driven by strong enhancers and active promoters.</a> Nucleic Acids Res. 42 (7), 4257-4269 (2014).</li><li>Gupta, S. 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=Bromo-+and+extraterminal+domain+chromatin+regulators+serve+as+cofactors+for+murine+leukemia+virus+integration.">Bromo- and extraterminal domain chromatin regulators serve as cofactors for murine leukemia virus integration.</a> J Virol. 87 (23), 12721-12736 (2013).</li><li>Sharma, 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=BET+proteins+promote+efficient+murine+leukemia+virus+integration+at+transcription+start+sites.">BET proteins promote efficient murine leukemia virus integration at transcription start sites.</a> Proc Natl Acad Sci U S A. 110 (29), 12036-12041 (2013).</li><li>De Rijck, 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=The+BET+family+of+proteins+targets+moloney+murine+leukemia+virus+integration+near+transcription+start+sites.">The BET family of proteins targets moloney murine leukemia virus integration near transcription start sites.</a> Cell reports. 5 (4), 886-894 (2013).</li><li>Romano, O., 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=Transcriptional,+epigenetic+and+retroviral+signatures+identify+regulatory+regions+involved+in+hematopoietic+lineage+commitment.">Transcriptional, epigenetic and retroviral signatures identify regulatory regions involved in hematopoietic lineage commitment.</a> Sci Rep. 6, 24724 (2016).</li><li>Cavazza, 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=Dynamic+Transcriptional+and+Epigenetic+Regulation+of+Human+Epidermal+Keratinocyte+Differentiation.">Dynamic Transcriptional and Epigenetic Regulation of Human Epidermal Keratinocyte Differentiation.</a> Stem Cell Reports. 6 (4), 618-632 (2016).</li><li>Miller, A. D., Law, M. F., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+helper-free+amphotropic+retroviruses+that+transduce+a+dominant-acting,+methotrexate-resistant+dihydrofolate+reductase+gene.">Generation of helper-free amphotropic retroviruses that transduce a dominant-acting, methotrexate-resistant dihydrofolate reductase gene.</a> Mol Cell Biol. 5 (3), 431-437 (1985).</li><li>Cattoglio, 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=High-definition+mapping+of+retroviral+integration+sites+defines+the+fate+of+allogeneic+T+cells+after+donor+lymphocyte+infusion.">High-definition mapping of retroviral integration sites defines the fate of allogeneic T cells after donor lymphocyte infusion.</a> PLoS One. 5 (12), e15688 (2010).</li><li>Aiuti, 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=Lentiviral+hematopoietic+stem+cell+gene+therapy+in+patients+with+Wiskott-Aldrich+syndrome.">Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome.</a> Science. 341 (6148), 1233151 (2013).</li><li>Biffi, 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=Lentiviral+hematopoietic+stem+cell+gene+therapy+benefits+metachromatic+leukodystrophy.">Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy.</a> Science. 341 (6148), 1233158 (2013).</li><li>Gabriel, 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=Comprehensive+genomic+access+to+vector+integration+in+clinical+gene+therapy.">Comprehensive genomic access to vector integration in clinical gene therapy.</a> Nat Med. 15 (12), 1431-1436 (2009).</li><li>Gillet, N. 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=The+host+genomic+environment+of+the+provirus+determines+the+abundance+of+HTLV-1-infected+T-cell+clones.">The host genomic environment of the provirus determines the abundance of HTLV-1-infected T-cell clones.</a> Blood. 117 (11), 3113-3122 (2011).</li></ol>

作者没有什么可以披露的。

在这里，我们描述了一个针对染色质区域的逆转录病毒mLV的整合位点的全基因组映射方案，表观遗传标记为活性启动子和增强子。方案的关键步骤和/或限制包括:（i）目标细胞群的mLV转导; （ii）通过LM-PCR扩增病毒宿主结; （iii）检索高比例的整合站点。基于mLV的逆转录病毒载体有效转导分裂细胞。非分裂细胞（例如有丝分裂后神经元细胞）的转导效率低是该技术的潜在限制。然而，可以通过基于?...

细胞同一性由特定基因组的表达决定。顺式调节元件（如启动子和增强子）的作用对于细胞型特异性转录程序的激活至关重要。这些调节区域的特征在于特定的染色质特征，例如特异性组蛋白修饰，转录因子和辅因子结合以及染色质可及性，其已被广泛用于在几种细胞类型1,2,3中的全基因组鉴定。特别地，组蛋白H3赖氨酸27（H3K27ac）的乙酰化的全基因组谱通常用于定义活性启动子，增强子和超增强子4,5,6 。 莫洛尼鼠白血病病毒（MLV）是广泛用于基因的γ-逆转录病毒转移哺乳动物细胞。感染靶细胞后，逆转录病毒RNA基因组被逆转录在双链DNA分子中，其结合病毒和细胞蛋白以组装前整合复合物（PIC）。 PIC进入核并结合宿主细胞染色质。在这里，病毒整合酶，一个关键的PIC组件，介导前病毒DNA整合到宿主细胞基因组。基因组DNA中的mLV整合不是随机的，而是发生在活性顺式调节元件（如启动子和增强子）中，细胞特异性方式为7,8,9,10 。这种特异的整合特征是通过mLV整合酶和细胞溴结构域和外切域（BET）蛋白质11,12,13之间的直接相互作用来介导的。 BET蛋白（BRD2，BRD3，BRD4）作为宿主染色质和mLV PIC之间的桥梁:通过它们的溴结构域，它们识别高度乙酰化的顺式调节区，而外发区与mLV整合酶11,12,13相互作用。 在这里，我们描述了逆转录病毒扫描，一种基于mLV的整合性质绘制活性顺式调节区的新工具。简言之，用表达增强的绿色荧光蛋白（eGFP）报道基因的来自mLV的逆转录病毒载体转导细胞。在基因组DNA提取后，通过连接物介导的PCR（LM-PCR）扩增mLV载体的3&#39;末端重复序列（LTR）和基因组DNA之间的连接并进行大规模测序。 mLV整合位点被映射到人类基因组，并且由mLV高度靶向的基因组区域被定义为mLV整合位点的簇。 逆转录病毒扫描ning用于在几个人类原代细胞14,15 中定义细胞特异性活性调节元件。 mLV簇与表观遗传学定义的启动子和增强子共同定位，其中大部分含有活性组蛋白标记，如H3K27ac，并且是细胞特异性的。逆转录病毒扫描允许在前瞻性纯化的细胞群体中的DNA调节元件的全基因组鉴定7,14以及缺乏有效标记物的回顾性定义的细胞群（例如角质形成细胞干细胞） 15 。

<table><tbody><tr><td>PBS, pH 7.4</td><td>ThermoScientific</td><td>10010031</td><td>or equivalent</td></tr><tr><td>Fetal Bovine Serum</td><td>ThermoScientific</td><td>16000044</td><td>or equivalent</td></tr><tr><td>0.2 ml tubes</td><td>general lab supplier</td><td></td><td></td></tr><tr><td>1.5 ml tubes</td><td>general lab supplier</td><td></td><td></td></tr><tr><td>QIAGEN QIAmp DNA mini Kit&amp;nbsp;</td><td>QIAGEN</td><td>51306</td><td>or equivalent</td></tr><tr><td>T4 DNA ligase&amp;nbsp;</td><td>New England BioLabs</td><td>M0202T</td><td></td></tr><tr><td>T4 DNA Ligase Reaction buffer</td><td>New England BioLabs</td><td>M0202T</td><td></td></tr><tr><td>Linker Plus Strand oligonucleotide</td><td>general lab supplier</td><td></td><td>5&amp;rsquo;-PO&lt;sub&gt;4&lt;/sub&gt;-TAGTCCCTTAAGCGGAG-3&amp;rsquo;&amp;nbsp; (Purification grade: SDS-PAGE)</td></tr><tr><td>Linker Minus Strand oligonucleotide</td><td>general lab supplier</td><td></td><td>5&amp;rsquo;-GTAATACGACTCACTATAGGGCTCCGCTTAAGGGAC-3&amp;rsquo; (Purification grade: SDS-PAGE)</td></tr><tr><td>Tru9I</td><td>Roche-Sigma-Aldrich</td><td>11464825001</td><td></td></tr><tr><td>SuRE/Cut Buffer M</td><td>Roche-Sigma-Aldrich</td><td>11417983001</td><td></td></tr><tr><td>PstI&amp;nbsp;</td><td>Roche-Sigma-Aldrich</td><td>10798991001</td><td></td></tr><tr><td>SuRE/Cut Buffer H</td><td>Roche-Sigma-Aldrich</td><td>11417991001</td><td></td></tr><tr><td>Platinum Taq DNA Polimerase High Fidelity&amp;nbsp;</td><td>Invitrogen</td><td>11304011</td><td></td></tr><tr><td>10 mM dNTP Mix</td><td>Invitrogen</td><td>18427013</td><td>or equivalent</td></tr><tr><td>PCR grade water</td><td>general lab supplier</td><td></td><td></td></tr><tr><td>96-well thermal cycler (with heated lid)</td><td>general lab supplier</td><td></td><td></td></tr><tr><td>linker primer</td><td>general lab supplier</td><td></td><td>5&amp;rsquo;-GTAATACGACTCACTATAGGGC-3&amp;rsquo; (Purification grade: PCR grade)</td></tr><tr><td>MLV-3&amp;rsquo; LTR primer</td><td>general lab supplier</td><td></td><td>5&amp;rsquo;-GACTTGTGGTCTCGCTGTTCCTTGG-3&amp;rsquo; (Purification grade: PCR grade)</td></tr><tr><td>linker nested primer 454</td><td>general lab supplier</td><td></td><td>5&amp;rsquo;-GCCTTGCCAGCCCGCTCAG[AGGGCTCCGCTTAAGGGAC](Purification grade: SDS-PAGE)</td></tr><tr><td>MLV-3&amp;rsquo; LTR nested primer 454</td><td>general lab supplier</td><td></td><td>5&amp;rsquo;-GCCTCCCTCGCGCCATCAGTAGC[GGTCTCCTCTGAGTGATTGACTACC](Purification grade: SDS-PAGE)</td></tr><tr><td>linker nested primer Illumina</td><td>general lab supplier</td><td></td><td>5&#039;-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-[AGGGCTCCGCTTAAGGGAC](Purification grade: SDS-PAGE)</td></tr><tr><td>MLV-3&amp;rsquo; LTR nested primer Illumina</td><td>general lab supplier</td><td></td><td>5&#039;-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-[GGTCTCCTCTGAGTGATTGACTACC](Purification grade: SDS-PAGE)</td></tr><tr><td>Sodium Acetate Solution (3M) pH 5.2</td><td>general lab supplier</td><td></td><td></td></tr><tr><td>Ethanol (absolute) for molecular biology</td><td>Sigma-Aldrich</td><td>E7023</td><td>or equivalent</td></tr><tr><td>Topo TA Cloning kit (with pCR2.1-TOPO vector)</td><td>Invitrogen</td><td>K4500-01</td><td></td></tr><tr><td>QIAquick Gel Extraction kit</td><td>QIAGEN</td><td>28704</td><td></td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A9539</td><td>or equivalent</td></tr><tr><td>Ethidium bromide&amp;nbsp;</td><td>Sigma-Aldrich</td><td>E1510</td><td>or equivalent</td></tr><tr><td>100 bp DNA ladder</td><td>Invitrogen</td><td>15628019</td><td>or equivalent</td></tr><tr><td>6x Loading Buffer</td><td>ThermoScientific</td><td>R0611</td><td>or equivalent</td></tr><tr><td>NanoDrop 2000 UV-Vis Spectrophotometer</td><td>ThermoScientific</td><td>ND-2000</td><td></td></tr><tr><td>Nextera XT Index kit</td><td>Illumina</td><td>FC-131-1001 or FC-131-1002</td><td></td></tr><tr><td>2x KAPA HiFi Hot Start Ready Mix&amp;nbsp;</td><td>KAPA Biosystems</td><td>KK2601</td><td></td></tr><tr><td>Dynal magnetic stand for 2 ml tubes</td><td>Invitrogen</td><td>12321D</td><td>or equivalent</td></tr><tr><td>Agencourt AMPure XP 60 ml kit</td><td>Beckman Coulter Genomics</td><td>A63881</td><td></td></tr><tr><td>Tris-HCl 10 mM, pH 8.5</td><td>general lab supplier</td><td></td><td></td></tr><tr><td>Agilent 2200 TapeStation system</td><td>Agilent Technologies</td><td>G2964AA</td><td>or equivalent</td></tr><tr><td>D1000 ScreenTape</td><td>Agilent Technologies</td><td>5067-5582</td><td>or equivalent</td></tr><tr><td>D1000 Reagents</td><td>Agilent Technologies</td><td>5067-5583</td><td>or equivalent</td></tr><tr><td>KAPA Library Quantification Kit for Illumina platforms (ABI Prism)</td><td>KAPA Biosystems</td><td>KK4835</td><td></td></tr><tr><td>ABI Prism 7900HT Fast Real-Time PCR System</td><td>Applied Biosystems</td><td>4329003</td><td></td></tr><tr><td>NaOH 1.0 N, molecular biology-grade</td><td>general lab supplier</td><td></td><td></td></tr><tr><td>HT1 (Hybridization Buffer)</td><td>Illumina&amp;nbsp;</td><td>Provided in the MiSeq Reagent Kit</td><td></td></tr><tr><td>MiSeq Reagent Kit v3 (150 cycles)</td><td>Illumina</td><td>MS-102-3001</td><td></td></tr><tr><td>MiSeq System</td><td>Illumina</td><td>SY-410-1003</td><td></td></tr><tr><td>PhiX Control v3</td><td>Illumina</td><td>FC-110-3001</td><td></td></tr></tbody></table>

retroviral scanning: mapping mlv integration sites to define cell-specific regulatory regions

莫洛尼鼠白血病（MLV）病毒的逆转录病毒载体主要集中在乙酰化增强子和启动子中。因此，mLV整合位点可用作活性调节元件的功能标记。在这里，我们提出了一种逆转录病毒扫描工具，可以在全基因组范围内鉴定细胞特异性增强子和启动子。简言之，用mLV衍生的载体转导靶细胞群，并用经常切割的限制酶消化基因组DNA。在用相容的DNA接头连接基因组片段后，连接子介导的聚合酶链反应（LM-PCR）允许扩增病毒宿主基因组连接。扩增子的大规模测序用于在全基因组范围内定义mLV整合谱。最后，定义了复发整合的集群，以确定细胞特异性调节区域，负责细胞型特异性转录程序的激活。 例如体细胞干细胞）的工具技术，其缺乏用于预期分离的鲁棒标记物。

逆转录病毒扫描程序的工作流程逆转录病毒扫描程序的工作流程如图1所示 。用表达eGFP报告基因的mLV衍生的逆转录病毒载体纯化并转导靶细胞群。转基因侧翼是两个相同的长末端重复序列（5&#39;和3&#39;LTR），确保病毒基因组合成，逆转录和整合到宿主DNA中。通过FACS分析eGFP表达来评估转导效率。扩增含有高?...

Watch this Scientific Journal Video about Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions at JoVE.com

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

在这里，我们描述了人类细胞中基于Moloney小鼠白血病病毒的逆转录病毒载体的整合位点全基因组图谱的方案。

逆转录病毒扫描:映射MLV集成站点以定义细胞特异性监管区域

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

retroviral-scanning-mapping-mlv-integration-sites-to-define-cell

Research

JoVE Journal

Genetics

8.4K 次观看.  University of Modena and Reggio Emilia. 在这里，我们描述了人类细胞中基于Moloney小鼠白血病病毒的逆转录病毒载体的整合位点全基因组图谱的方案。 

视频: Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

双向逆转录病毒整合位点PCR方法和定量数据分析工作流程

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

科研

遗传学

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

用于下一代测序的近全长 HIV-1 Proviruses 的扩增

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

应用生物标志物发现在前列腺癌：个别赫尔维位点表达微阵列鉴定

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

医学

InSET: A Novel Tool for Unbiased Genome-Wide Functional Element Discovery

Identification of Functionally-Relevant Lentivirus Integration Sites in an Insertional Mutagenesis Cell Library

The extent of functional sequences within the human genome is a pivotal yet debated topic in biology. Although high-throughput reverse genetic screens have made strides in exploring this, they often limit their scope to known genomic elements and may introduce non-specific effects. This underscores the urgent need for novel functional genomics tools that enable a deeper, unbiased understanding of genome functionality. This protocol introduces the Insertion-based Screen for functional Elements and Transcripts (InSET), a method for identifying lentivirus integration sites within a lentivirus-based insertional mutagenesis cell library. InSET facilitates the capture of genome-wide lentiviral integration sites, with next-generation sequencing used to detect and quantify flanking sequences. InSET's design enables the analysis of integration site abundance variations in phenotypic screens on a large scale, establishing it as a robust tool for forward genetics and for identifying functional genomic elements. A key benefit of InSET is its capacity to reveal previously unidentified genomic elements, including novel functional exons of both protein-coding and non-coding RNAs, independent of prior annotation. Overall, InSET holds significant value in studying the intricate complexity of the human genome and transcriptome, where many genomic elements await functional characterization.

Insertional mutagenesis is an essential tool in forward genetics for identifying functional genomic elements. Here, we describe the Insertion-based Screen for functional Elements and Transcripts (InSET), a method for detecting lentivirus integration sites within a lentivirus-based insertional mutagenesis cell library.

The extent of functional sequences within the human genome is a pivotal yet debated topic in biology. Although high-throughput reverse genetic screens ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

虫媒病毒感染作为鉴定病毒免疫调节剂和宿主抗病毒因子的筛选工具

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

免疫与感染

CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites

Human immunodeficiency virus (HIV) integrates its proviral DNA non-randomly into the host cell genome at recurrent sites and genomic hotspots. Here we present a detailed protocol for the generation of novel in vitro models for HIV infection with chosen genomic integration sites using CRISPR-Cas9-based genome engineering technology. With this method, a reporter sequence of choice can be integrated into a targeted, chosen genomic locus, reflecting clinically relevant integration sites.
In the protocol, the design of an HIV-derived reporter and choosing of a target site and gRNA sequence are described. A targeting vector with homology arms is constructed and transfected into Jurkat T cells. The reporter sequence is targeted to the selected genomic site by homologous recombination facilitated by a Cas9-mediated double-strand break at the target site. Single-cell clones are generated and screened for targeting events by flow cytometry and PCR. Selected clones are then expanded, and correct targeting is verified by PCR, sequencing, and Southern blotting. Potential off-target events of CRISPR-Cas9-mediated genome engineering are analyzed.
By using this protocol, novel cell culture systems that model HIV infection at clinically relevant integration sites can be generated. Although the generation of single-cell clones and verification of correct reporter sequence integration is time-consuming, the resulting clonal lines are powerful tools to functionally analyze proviral integration site choice.

We present a genome engineering workflow for the generation of new in vitro models for HIV-1 infection that recapitulate proviral integration at selected genomic sites. Targeting of HIV-derived reporters is facilitated by CRISPR-Cas9-mediated, site-specific genome manipulation. Detailed protocols for single-cell clone generation, screening, and correct targeting verification are provided.

基于 crispr-cas9 的基因组工程, 用于生成 hiv-1 感染与选定的证明病毒整合点的 jurkat 报告模型

Human immunodeficiency virus (HIV) integrates its proviral DNA non-randomly into the host cell genome at recurrent sites and genomic hotspots. Here we ...

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune system and is not targeted by the current antiretroviral therapy (cART). Transcription and splicing have been shown to reinforce HIV-1 latency in resting CD4+ T cells. Reversal of latency by the use of latency reversal agents (LRAs) in the &#34;shock and kill&#34; approach has been studied extensively in an attempt to purge this reservoir but has thus far not shown any success in clinical trials due to the lack of development of adequate small molecules that can efficiently perturb this reservoir. The protocol presented here provides a method for reliably and efficiently assessing latency reversal agents (LRAs) on HIV transcription and splicing. This approach is based on the use of an LTR-driven dual color reporter that can simultaneously measure the effect of an LRA on transcription and splicing by flow cytometry. The protocol described here is adequate for adherent cells as well as the cells in suspension. It is useful for testing a large number of drugs in a high throughput system. The method is technically simple to implement and cost-effective. In addition, the use of flow cytometry allows the assessment of cell viability and thus drug toxicity at the same time.

A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided.

hiv 转录和拼接中延迟逆转剂的高通量体外评价

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune ...

Linear Amplification Mediated PCR &#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with integrating vector systems. Due to the stable integration of retroviral vectors, integration sites can be used to study the clonal fate of individual cells and their progeny. LAM- PCR for the first time provided evidence that leukemia in gene therapy treated patients originated from provirus induced overexpression of a neighboring proto-oncogene. The high sensitivity and specificity of LAM-PCR compared to existing methods like inverse PCR and ligation mediated (LM)-PCR is achieved by an initial preamplification step (linear PCR of 100 cycles) using biotinylated vector specific primers which allow subsequent reaction steps to be carried out on solid phase (magnetic beads). LAM-PCR is currently the most sensitive method available to identify&#160;unknown DNA which is located in the proximity of known DNA. Recently, a variant of LAM-PCR has been developed that circumvents restriction digest thus abrogating retrieval bias of integration sites and enables a comprehensive analysis of provirus locations in host genomes. The following protocol explains step-by-step the amplification of both 3&#8217;- and 5&#8217;- sequences adjacent to the integrated lentiviral vector.

Linear-amplification mediated (LAM)-PCR is a method developed to identify the exact positions of integrating viral vectors in the genome. The technique has evolved to be the superior method to study clonal dynamics in gene therapy patients, biosafety of novel vector technologies, T-cell diversity, cancer stem cell models, etc.

线性扩增介导的PCR  - 未知侧翼DNA的遗传物质和表征本地化

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with ...

生物学

破译急性 LCMV 感染中的早期滤泡辅助性 T 细胞定型术

Accessing Early Differentiation of Virus-Specific Follicular Helper CD4+ T Cell in Acute LCMV-Infected Mice

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, thus establishing long-term humoral immunity. During acute viral infection, the fate commitment of virus-specific TFH cells is determined in the early infection phase, and investigations of the early-differentiated TFH cells are crucial in understanding T cell-dependent humoral immunity and optimizing vaccine design. In the study, using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection and the TCR-transgenic SMARTA (SM) mouse with CD4+ T cells specifically recognizing LCMV glycoprotein epitope I-AbGP66-77, we described procedures to access the early fate commitment of virus-specific TFH cells based on flow cytometry stainings. Furthermore, by exploiting retroviral transduction of SM CD4+ T cells, methods to manipulate gene expression in early-differentiated virus-specific TFH cells are also provided. Hence, these methods will help in studies exploring the mechanism(s) underlying the early commitment of virus-specific TFH cells.

作者聚焦:阐明急性 LCMV 挑战中 TFH 细胞分化的途径

The current study showcases protocols for assessing the early fate commitment of virus-specific TFH cells and manipulating gene expression in these cells.

在急性 LCMV 感染小鼠中获得病毒特异性滤泡辅助性 CD4 + T 细胞的早期分化

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, ...

逆转录病毒扫描:映射MLV集成站点以定义细胞特异性监管区域

摘要

探索更多视频

此视频中的章节