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
Summary
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。
Abstract
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。
<p class=“jove_content”>逆转录病毒扫描工具可以在前瞻性分离的靶细胞群体中全基因组鉴定细胞特异性启动子和增强子。值得注意的是，逆转录病毒扫描是用于回顾性鉴定稀有群体（ 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。Introduction
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'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 。
Protocol
人类细胞的MLV转导 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。 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…
Representative Results
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'和3'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?…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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）。
Materials
| PBS, pH 7.4 | ThermoScientific | 10010031 | or equivalent |
| Fetal Bovine Serum | ThermoScientific | 16000044 | or equivalent |
| 0.2 ml tubes | general lab supplier | ||
| 1.5 ml tubes | general lab supplier | ||
| QIAGEN QIAmp DNA mini Kit | QIAGEN | 51306 | or equivalent |
| T4 DNA ligase | New England BioLabs | M0202T | |
| T4 DNA Ligase Reaction buffer | New England BioLabs | M0202T | |
| Linker Plus Strand oligonucleotide | general lab supplier | 5’-PO4-TAGTCCCTTAAGCGGAG-3’ (Purification grade: SDS-PAGE) | |
| Linker Minus Strand oligonucleotide | general lab supplier | 5’-GTAATACGACTCACTATAGGGCTCCGCTTAAGGGAC-3’ (Purification grade: SDS-PAGE) | |
| Tru9I | Roche-Sigma-Aldrich | 11464825001 | |
| SuRE/Cut Buffer M | Roche-Sigma-Aldrich | 11417983001 | |
| PstI | Roche-Sigma-Aldrich | 10798991001 | |
| SuRE/Cut Buffer H | Roche-Sigma-Aldrich | 11417991001 | |
| Platinum Taq DNA Polimerase High Fidelity | Invitrogen | 11304011 | |
| 10mM dNTP Mix | Invitrogen | 18427013 | or equivalent |
| PCR grade water | general lab supplier | ||
| 96-well thermal cycler (with heated lid) | general lab supplier | ||
| linker primer | general lab supplier | 5’-GTAATACGACTCACTATAGGGC-3’ (Purification grade: PCR grade) | |
| MLV-3’ LTR primer | general lab supplier | 5’-GACTTGTGGTCTCGCTGTTCCTTGG-3’ (Purification grade: PCR grade) | |
| linker nested primer 454 | general lab supplier | 5’-GCCTTGCCAGCCCGCTCAG[AGGGCTCCGCTTAAGGGAC](Purification grade: SDS-PAGE) | |
| MLV-3’ LTR nested primer 454 | general lab supplier | 5’-GCCTCCCTCGCGCCATCAGTAGC[GGTCTCCTCTGAGTGATTGACTACC](Purification grade: SDS-PAGE) | |
| linker nested primer Illumina | general lab supplier | 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-[AGGGCTCCGCTTAAGGGAC](Purification grade: SDS-PAGE) | |
| MLV-3’ LTR nested primer Illumina | general lab supplier | 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-[GGTCTCCTCTGAGTGATTGACTACC](Purification grade: SDS-PAGE) | |
| Sodium Acetate Solution (3M) pH 5.2 | general lab supplier | ||
| Ethanol (absolute) for molecular biology | Sigma-Aldrich | E7023 | or equivalent |
| Topo TA Cloning kit (with pCR2.1-TOPO vector) | Invitrogen | K4500-01 | |
| QIAquick Gel Extraction kit | QIAGEN | 28704 | |
| Agarose | Sigma-Aldrich | A9539 | or equivalent |
| Ethidium bromide | Sigma-Aldrich | E1510 | or equivalent |
| 100 bp DNA ladder | Invitrogen | 15628019 | or equivalent |
| 6X Loading Buffer | ThermoScientific | R0611 | or equivalent |
| NanoDrop 2000 UV-Vis Spectrophotometer | ThermoScientific | ND-2000 | |
| Nextera XT Index kit | Illumina | FC-131-1001 or FC-131-1002 | |
| 2x KAPA HiFi Hot Start Ready Mix | KAPA Biosystems | KK2601 | |
| Dynal magnetic stand for 2 ml tubes | Invitrogen | 12321D | or equivalent |
| Agencourt AMPure XP 60 ml kit | Beckman Coulter Genomics | A63881 | |
| Tris-HCl 10 mM, pH 8.5 | general lab supplier | ||
| Agilent 2200 TapeStation system | Agilent Technologies | G2964AA | or equivalent |
| D1000 ScreenTape | Agilent Technologies | 5067-5582 | or equivalent |
| D1000 Reagents | Agilent Technologies | 5067-5583 | or equivalent |
| KAPA Library Quantification Kit for Illumina platforms (ABI Prism) | KAPA Biosystems | KK4835 | |
| ABI Prism 7900HT Fast Real-Time PCR System | Applied Biosystems | 4329003 | |
| NaOH 1.0 N, molecular biology-grade | general lab supplier | ||
| HT1 (Hybridization Buffer) | Illumina | Provided in the MiSeq Reagent Kit | |
| MiSeq Reagent Kit v3 (150 cycles) | Illumina | MS-102-3001 | |
| MiSeq System | Illumina | SY-410-1003 | |
| PhiX Control v3 | Illumina | FC-110-3001 |
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Cite This Article
Romano, O., Cifola, I., Poletti, V., Severgnini, M., Peano, C., De Bellis, G., Mavilio, F., Miccio, A. Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions. J. Vis. Exp. (123), e55919, doi:10.3791/55919 (2017).