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>

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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&#160;</td>
			<td>QIAGEN</td>
			<td>51306</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>T4 DNA ligase&#160;</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&#8217;-PO4-TAGTCCCTTAAGCGGAG-3&#8217;&#160; (Purification grade: SDS-PAGE)</td>
		</tr>
		<tr>
			<td>Linker Minus Strand oligonucleotide</td>
			<td>general lab supplier</td>
			<td></td>
			<td>5&#8217;-GTAATACGACTCACTATAGGGCTCCGCTTAAGGGAC-3&#8217; (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&#160;</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&#160;</td>
			<td>Invitrogen</td>
			<td>11304011</td>
			<td></td>
		</tr>
		<tr>
			<td>10mM 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&#8217;-GTAATACGACTCACTATAGGGC-3&#8217; (Purification grade: PCR grade)</td>
		</tr>
		<tr>
			<td>MLV-3&#8217; LTR primer</td>
			<td>general lab supplier</td>
			<td></td>
			<td>5&#8217;-GACTTGTGGTCTCGCTGTTCCTTGG-3&#8217; (Purification grade: PCR grade)</td>
		</tr>
		<tr>
			<td>linker nested primer 454</td>
			<td>general lab supplier</td>
			<td></td>
			<td>5&#8217;-GCCTTGCCAGCCCGCTCAG[AGGGCTCCGCTTAAGGGAC](Purification grade: SDS-PAGE)</td>
		</tr>
		<tr>
			<td>MLV-3&#8217; LTR nested primer 454</td>
			<td>general lab supplier</td>
			<td></td>
			<td>5&#8217;-GCCTCCCTCGCGCCATCAGTAGC[GGTCTCCTCTGAGTGATTGACTACC](Purification grade: SDS-PAGE)</td>
		</tr>
		<tr>
			<td>linker nested primer Illumina</td>
			<td>general lab supplier</td>
			<td></td>
			<td>5&#39;-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-[AGGGCTCCGCTTAAGGGAC](Purification grade: SDS-PAGE)</td>
		</tr>
		<tr>
			<td>MLV-3&#8217; LTR nested primer Illumina</td>
			<td>general lab supplier</td>
			<td></td>
			<td>5&#39;-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&#160;</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&#160;</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&#160;</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

8.3K 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

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites.

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Details are presented on how QTL mapping with a whole genome sequence based genetic map can be used to identify a drug resistance gene in Toxoplasma gondii and how this can be verified with the CRISPR/Cas9 system that efficiently edits a genomic target, in this case the drug resistance gene.

QTL定位和CRISPR / Cas9编辑识别抗药性基因<em&gt;弓形虫</em

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the ...

科研

遗传学

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

使用生物素化补骨脂素全球定位RNA-RNA相互作用

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

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 ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

CRISPR/Cas9-mediated 目标集成在体内使用基于同源介导的端接连接策略

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

The plasticity of nuclear architecture is controlled by dynamic epigenetic mechanisms including non-coding RNAs, DNA methylation, nucleosome repositioning as well as histone composition and modification. Here we describe a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) protocol which allows the determination of chromatin accessibility in a reproducible, inexpensive, and straightforward manner.

甲醛辅助分离测定哺乳动物细胞染色质可及性的调控元素

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly being extended to integrative studies to enhance the understanding of molecular dynamics within cells. Current tools for the visualization and integration of proteomics with other omics datasets are inadequate for large-scale studies. Furthermore, they only capture basic sequence identify, discarding post-translational modifications and quantitation. To address these issues, we developed PoGo to map peptides with associated post-translational modifications and quantification to reference genome annotation. In addition, the tool was developed to enable the mapping of peptides identified from customized sequence databases incorporating single amino acid variants. While PoGo is a command line tool, the graphical interface PoGoGUI enables non-bioinformatics researchers to easily map peptides to 25 species supported by Ensembl genome annotation. The generated output borrows file formats from the genomics field and, therefore, visualization is supported in most genome browsers. For large-scale studies, PoGo is supported by TrackHubGenerator to create web-accessible repositories of data mapped to genomes that also enable an easy sharing of proteogenomics data. With little effort, this tool can map millions of peptides to reference genomes within only a few minutes, outperforming other available sequence-identity based tools. This protocol demonstrates the best approaches for proteogenomics mapping through PoGo with publicly available datasets of quantitative and phosphoproteomics, as well as large-scale studies.

Here we present the proteogenomic tool PoGo and protocols for fast, quantitative, post-translational modification and variant enabled mapping of peptides identified through mass spectrometry onto reference genomes. This tool is of use to integrate and visualize proteogenomic and personal proteomic studies interfacing with orthogonal genomics data.

一种快速、定量的后向基因组转化和变异的多肽映射方法

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly ...

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to control gene expression. These regulatory proteins control gene expression either at the level of DNA (transcription) or at the level of RNA (pre-mRNA splicing, polyadenylation, mRNA transport, decay, and translation). Identification of regulatory sequences helps understand not only how a gene is switched on or off, but also which downstream genes are regulated by a particular regulatory protein. Here, we describe a one-step approach that allows saturation mutagenesis of a protein binding site in RNA. It involves doping DNA template with non-wild-type nucleotides within the binding site, synthesis of separate RNAs with each phosphorothioate nucleotide, and isolation of the bound fraction following incubation with protein. Interference from non-wild-type nucleotides results in their preferential exclusion from the protein-bound fraction. This is monitored by gel electrophoresis following selective chemical cleavage with iodine of phosphodiester bonds containing phosphorothioates (phosphorothioate mutagenesis or PTM). This single-step saturation mutagenesis approach is applicable to the characterization of any protein binding site in RNA.

Proteins that bind specific RNA sequences play critical roles in gene expression. Detailed characterization of these binding sites is crucial for our understanding of gene regulation. Here, a single-step approach for saturation mutagenesis of protein-binding sites in RNA is described. This approach is relevant for all protein-binding sites in RNA.

一种新的饱和诱变方法: 利用 Phosphorothioates 对 RNA 调控蛋白结合位点的单步表征

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to ...

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

单液滴数字聚合酶链反应对热点地区突变的综合和同步检测

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational steps are used to regulate specific genes. Sequence-specific nucleic acid-binding proteins target specific sequences to control spatial or temporal gene expression. The binding sites in nucleic acids are typically characterized by mutational analysis. However, numerous proteins of interest have no known binding site for such characterization. Here we describe an approach to identify previously unknown binding sites for RNA-binding proteins. It involves iterative selection and amplification of sequences starting with a randomized sequence pool. Following several rounds of these steps-transcription, binding, and amplification-the enriched sequences are sequenced to identify a preferred binding site(s). Success of this approach is monitored using in vitro binding assays. Subsequently, in vitro and in vivo functional assays can be used to assess the biological relevance of the selected sequences. This approach allows identification and characterization of a previously unknown binding site(s) for any RNA-binding protein for which an assay to separate protein-bound and unbound RNAs exists.

Sequence specificity is critical for gene regulation. Regulatory proteins that recognize specific sequences are important for gene regulation. Defining functional binding sites for such proteins is a challenging biological problem. An iterative approach for identification of a binding site for an RNA-binding protein is described here and is applicable to all RNA-binding proteins.

探索序列空间,识别调节RNA结合蛋白的结合位点

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational ...

Transcription Start Site Mapping Using Super-low Input Carrier-CAGE

Cap analysis of gene expression (CAGE) is a method used for single-nucleotide resolution detection of RNA polymerase II transcription start sites (TSSs). Accurate detection of TSSs enhances identification and discovery of core promoters. In addition, active enhancers can be detected through signatures of bidirectional transcription initiation. Described here is a protocol for performing super-low input carrier-CAGE (SLIC-CAGE). This SLIC adaptation of the CAGE protocol minimizes RNA losses by artificially increasing the RNA amount through use of an in vitro transcribed RNA carrier mix that is added to the sample of interest, thus enabling library preparation from nanogram-amounts of total RNA (i.e., thousands of cells). The carrier mimics the expected DNA library fragment length distribution, thereby eliminating biases that could be caused by the abundance of a homogenous carrier. In the last stages of the protocol, the carrier is removed through degradation with homing endonucleases and the target library is amplified. The target sample library is protected from degradation, as the homing endonuclease recognition sites are long (between 18 and 27 bp), making the probability of their existence in the eukaryotic genomes very low. The end result is a DNA library ready for next-generation sequencing. All steps in the protocol, up to sequencing, can be completed within 6 days. The carrier preparation requires a full working day; however, it can be prepared in large quantities and kept frozen at -80 &#176;C. Once sequenced, the reads can be processed to obtain genome-wide single-nucleotide resolution TSSs. TSSs can be used for core promoter or enhancer discovery, providing insight into gene regulation. Once aggregated to promoters, the data can also be used for 5&#8217;-centric expression profiling.

Cap Analysis of Gene Expression (CAGE) is a method for genome-wide quantitative mapping of mRNA 5&#8217;ends to capture RNA polymerase II transcription start sites at a single-nucleotide resolution. This work describes a low-input (SLIC-CAGE) protocol for generation of high-quality libraries using nanogram-amounts of total RNA.

使用超低输入载波保持架进行转录启动站点映射

Cap analysis of gene expression (CAGE) is a method used for single-nucleotide resolution detection of RNA polymerase II transcription start sites (TSSs). ...