pSicoR PGK Puro慢病毒载体使用说明

合集下载

慢病毒使用操作指南

慢使用操作指南慢使用操作指南1.慢简介1.1 定义：慢是一种特殊的，能够在细胞中长期存活并进行复制。

1.2 用途：慢广泛应用于基因转导、基因敲除和基因表达等实验研究中。

2.慢使用前准备工作2.1 实验室准备工作2.1.1 实验室空间准备：确保实验室内有足够的操作空间和合适的消毒设备。

2.1.2 材料准备：准备好所需的培养基、细胞培养物、慢载体等。

2.1.3 设备准备：确保离心机、冰箱等设备正常工作，并准备好相关的仪器和器材。

2.2 人员准备工作2.2.1 人员培训：对参与慢操作的人员进行培训，了解操作步骤和安全风险。

2.2.2 个人防护：提供必要的防护装备，如实验服、手套、面罩等。

3.慢操作步骤3.1 细胞培养3.1.1 细胞培养物准备：根据实验需求选择合适的细胞培养物，并进行细胞的预处理和培养。

3.1.2 细胞密度调整：根据实验要求，调整细胞培养物的密度，以保证细胞的正常生长。

3.2 慢感染3.2.1 慢载体注射：将慢载体注射到培养好的细胞中。

3.2.2 感染条件控制：根据实验需求，控制慢感染的时间、浓度和温度等条件。

3.3 细胞培养和检测3.3.1 细胞培养：将感染好的细胞进行培养，并观察细胞的生长状态。

3.3.2 细胞检测：使用相关实验方法，对感染细胞进行检测和分析。

4.实验安全措施4.1 操作环境控制：确保实验室内通风良好，避免慢的扩散和污染。

4.2 废液处理：将产生的废液经过正确处理，避免对环境和人体造成污染和伤害。

4.3 事故应急处理：在发生事故或意外情况时，立即采取应急措施，并及时报告相关人员。

5.附件本文档所涉及的附件，包括但不限于实验记录表格、实验数据文件等。

6.法律名词及注释6.1 慢：指一种具有长周期和潜伏期的。

7.结束语感谢您阅读本文档，如有任何疑问或意见，请随时与我们联系。

2020年整理)慢病毒稳转细胞株步骤

作者：空青山作品编号：89964445889663Gd53022257782215002时间：2020.12.13稳转慢病毒一、所需试剂1、慢病毒载体（详细信息见附录及《质粒的扩增提取》）（大肠杆菌-80℃保存2-3年，质粒-20℃保存2-3年，病毒液-80℃保存1年）（1）载体质粒：两端的LTR、剪切位点、包装信号Ψ以及抗性或荧光基因、gag基因5′端350bp的序列及位于env序列中的RRE，含宿主RNA聚合酶识别部分（2）包装质粒（psPAX2）：包含了pol、gag包装成分（3）包膜质粒（pMD2.G）：用其他病毒的包膜蛋白代替了env基因.三种质粒共同转染产生不具有自我复制能力的病毒载体。

2、包装细胞：293T细胞3、菌株：大肠杆菌，用于提取质粒4、转染试剂：XTREME-GENE（-20℃保存，不可分装），一种脂质与其他组份构成的混合物5、浓缩试剂（配好后4℃保存，原材料室温保存）：5X PEG8000/NaCl溶液（聚乙二醇）：NaCl 8.766 g; PEG8000 50g溶解在200ml Milli-Q纯水中，高压蒸汽灭菌**也可直接从公司买来病毒液（-80℃封口膜封口冻存管保存，4℃保存3天）：滴度一般为108TU/ml6、10mg/ml polybrene（-20℃分装保存）：溴化己二甲铵。

是带正电的小分子，与细胞表面的阴离子结合，提高慢病毒对细胞的感染效率，通常加入polybrene 能提高感染效率2～10 倍。

有一定细胞毒性，需要摸索浓度（1～10μg/ml）7、无血清培养基：optimen8、贴壁细胞（复苏后3代以上的细胞）9、puromycin：嘌呤霉素，用于筛选稳转细胞二、具体步骤<一>病毒包装与收集（中皿，转染步骤类似于瞬转）第一天1、种板，10×105个293T细胞，加入全培养基双抗DMEM 4-5ml，过夜2、配制5X PEG8000/NaCl溶液称取NaCl 8.766 g; PEG8000 50g溶解在200ml Milli-Q纯水中；121摄氏度 30min 湿热灭绝 30min；保存在4℃第二天2、加入2ml全培养基DMEM3、将1加入2，孵育10h，换成5ml全培养基第四天第五天：1、9:00和17:00各收取一次5ml培养液，共20ml（-80℃保存）2、过滤：用孔径为0.45mm的过滤器除去上清中的293T细胞3、加入5ml 5XPEG8000/NaCl溶液，每30min-1h上下摇匀一次4、4℃过夜第六天1、4℃，3500rpm，20min2、弃上清，倒扣纸上静置1-2min，吸干残余液体3、加入120-150μl PBS，缓慢吹打，以防形成气溶胶4、50μl分装，-80℃保存。

慢病毒包装和应用实践

慢病毒包装和应用实践目录第3代慢病毒包装载体转移质粒（表达载体）包装质粒包装质粒包装质粒需全新构建，在MCS （多克隆位点）加入目的序列为产生高滴度的病毒颗粒，需要利用转移质粒和包装质粒同时共转染细胞无需全新构建，一次提取后分装慢病毒包装转移质粒的结构介绍----对照质粒◆LTR：长末端重复序列（long terminal repeat）反转录病毒的基因组的两端各有一个长末端重复序列(5'—LTR和3'—LTR)，不编码蛋白质，但含有启动子，增强子等调控元件。

参与前病毒进入宿主基因组，调控病毒基因的表达。

◆Psi：衣壳包装序列◆RRE：Rev反应元件◆cPPT ：中央聚嘌呤—中央末端序列◆WPRE：突变的土拨鼠肝炎病毒转录后调控元件◆U3PPT：多聚腺苷酸化相关序列●Promoter：启动子序列●GFP：绿色荧光蛋白慢病毒包装转移质粒的结构介绍CMV：Cytomegalovirus promoter 来源于巨细胞病毒的启动子EF1α: 延伸因子1α启动子MCS：multiple cloning site 多克隆位点PGK：phosphoglycerate kinase 1 promoter 磷酸甘油酸激酶基因启动子Puro：嘌呤霉素抗性基因2A：自剪切肽IRES：内部核糖体进入位点序列（Internal ribosome entry site）。

慢病毒包装转移质粒的结构介绍----启动子慢病毒包装转移质粒的结构介绍----多克隆位点◆多克隆位点含有限制性内切酶切割位点◆可用相应的酶将环状载体线性化，形成粘性末端或平末端◆这些酶切位点在载体上一般有且只有一个◆传统的载体构建方法需要依赖于对这些位点的切割IRES：(internal ribosomal entry sites)内部核糖体结合位点：一段较短的RNA序列（约150-250BP），这类RNA序列能折叠成类似于起始tRNA的结构，从而介导核糖体与RNA结合，起始蛋白质翻译。

pLenti6.3-MCS慢病毒载体使用说明

pLenti6.3-MCS慢病毒载体使⽤说明pLenti6.3-MCSpLenti6.3-MCS载体基本信息：载体名称: pLenti6.3-MCS质粒类型: 慢病毒表达载体⾼拷贝/低拷贝: --启动⼦: --克隆⽅法: 多克隆位点，限制性内切酶载体⼤⼩: --5' 测序引物及序列: --3' 测序引物及序列: --载体标签: --载体抗性: --筛选标记: --备注: --稳定性: --组成型: --病毒/⾮病毒: 慢病毒pLenti6.3-MCS载体质粒图谱和多克隆位点信息pLenti6.3-MCS载体序列pLenti6.3-MCS其他相关慢病毒载体：Tet-pLKO-neo Tet-pLKO-puro pPACKH1-GAGpMD2.G pCMV-dR8.2-dvpr pLKO.1-GFP-shRNA pLKO.1-TRC control pLKO.1-hygro pLKO.1-TRCpCDH-MSCV-MCS-EF1-copGFP pCDH-MSCV-MCS-EF1-copGFP-T2A-Puro FUW-tetO-hOKMSFUW-tetO-hOCT4 FUW-tetO-hSOX2 FUW-tetO-hKLF4FUW pLVX-AcGFP1-N1 pLVX-AcGFP1-C1pLVX-AmCyan1-N1 pLVX-DsRed-Express2-C1 pLVX-DsRed-Express2-N1 pLVX-DsRed-Monomer-N1 pLVX-PAmCherry-C1 pLVX-PAmCherry-N1 pLVX-ZsGreen1-N1 pLVX-IRES-ZsGreen1 pLVX-IRES-mCherry pLVX-mCherry-C1 pLVX-mCherry-N1 pLVX-tdTomato-C1 pLKO.1-puro pLentilox 3.7 pLVX-Tet-On-Advanced pLVX-IRES-Puro pLVX-IRES-Neo pLVX-IRES-HygpLVX-EF1α-DsRed-Monomer-C1 pLVX-EF1α-AcGFP1-N1 pLVX-EF1α-AcGFP1-C1 pLVX-EF1α-mCherry-C1 pLVX-EF1α-IRES-mCherry pLVX-EF1α-IRES-ZsGreen1 pLVX-MetLuc Control pLVX-MetLuc pLVX-Hom-Mem1pLVX-Het-2 pLVX-DD-AcGFP1-Actin pPRIME-TET-GFP-FF3pSIH1-H1-CopGFP pCDH-EF1-MCS-T2A-Puro pCDH-CMV-MCS-EF1-Puro pCDF1-MCS2-EF1-copGFP pLOX-CWBmi1 pLOX-CW-CREpRSV-rev pMDLg-pRRE pLL3.7pLVX-DD-AmCyan1 Control pLVX-DD-AmCyan1 Reporter pLVX-DD-tdTomato Reporter pLVX-DD-tdTomato Control pLVX-PTuner-Green pLVX-CherryPicker2pLVX-TetOne-Puro-Luc pLVX-TetOne pLVX-TetOne-PuropLVX-TetOne-Luc pLVX-rHom-Nuc1 pLVX-rHom-Sec1pLVX-rHom-1 pLVX-Hom-Nuc1 pLVX-Het-Nuc1pLVX-PTuner pLVX-PTuner2 pLVX-DD-ZsGreen1 Reporter pLVX-Het-1 pLVX-CherryPicker Control pLVX-Tet3GpCDH-CMV-MCS-EF1-RFP-T2A-Puro pCDH-CMV-MCS-EF1-Hygro pCDH-CMV-MCS-EF1-Neo pCDH-MCS-T2A-Puro-MSCV pCDH1-MCS2-EF1-copGFP pCDF1-MCS2-EF1-Puro pCDH-EF1-MCS-T2A-copGFP pWPXL pLVX-TRE3G-ZsGreen1 pLVX-TRE3G-mCherry pLenti6.3-EmGFP-BveI miR pLenti6/V5-GW/lacZpLenti6.3/V5-GW/EmGFP pLenti6.3-MCS pLenti6.3-DsRed2-BveI miR pLenti6.3-MCS-IRES2-EGFP pLVX-shRNA2 psPAX2VSV-G pSico PGK Puro pcDNA6.2-DsRed2-MCS1 miR pcDNA6.3-EmGFP-NC- II pcDNA6.2-EmGFP-NC- I pcDNA6.2-EmGFP-BsaI miR pLenti6.3-BveI miR pLenti6.3-MCS-IRES2-DsRed2 pLEX-MCSpGIPZ pLP2 pLP1FUGW pFUGW pLOX-Ttag-iresTKpMDLg/pRRE pLentG-KOSM pCMV-dR8.91pLVX-TRE3G-Luc Control pLVX-TRE3G-IRES pCgpvpSico pSicoR pLVTHMpGensil-1 pLVX-EF1α-IRES-Puro pCDF1-MCS2-EF1-copGFP pPACKH1-REV pLVX-Het-Mem1 pLVX-shRNA1pLKO.1-puro-GFP-siRNA pPRIME-TREX-GFP-FF3 pcDNA6.2-DsRed2-BsmBI miR pCDH-MSCV-MCS-EF1-Puro pCDH-CMV-MCS-EF1-copGFP pLVX-TRE3GFUW-tetO-hMYC pLOX-TERT-iresTK pLP/VSVGFUW-M2rtTA pCDH-EF1-MCS-(PGK-Puro) pcDNA6.2-EmGFP-MCS1 miR pLVX-AmCyan1-C1 pLVX-Hom-1 pcDNA6.2-BsaI miRpLVX-DsRed-Monomer-C1 pLVX-mCherry-Actin pTRIPZpLVX-ZsGreen1-C1 pLVX-CherryPicker1 LeGO-iC2pLVX-IRES-tdTomato pCDH-CMV-MCS-EF1-copGFP-T2A-Puro pLKO.3GpLVX-tdTomato-N1 pLVX-PTuner2-C pLVX-PuropLVX-Tight-Puro pLVX-DD-ZsGreen1 Control pSicoR PGK PuropLVX-EF1α-DsRed-Monomer-N1 pCDH-UbC-MCS-EF1-Hygro pLVTHpLVX-EF1α-mCherry-N1 pCDH-CMV-MCS-EF1-RFP。

整理慢病毒稳转细胞株步骤

整理慢病毒稳转细胞株步骤竹密岂妨流水过蛮鬼2021. 3山高哪碍野云飞稳转慢病毒一、所需试剂1、慢病毒载体（详细信息见附录及《质粒的扩增提取》）（大肠杆菌-80°C保存2-3 年，质粒-20°C保存2-3年，病毒液-80°C保存1年）（1）载体质粒：两端的LTR、剪切位点、包装信号屮以及抗性或荧光基因、gag基因5’端350bp的序列及位丁,nv序列中的RRE,含宿主RNA聚合酶识别部分（2）包装质粒（psPAX2）:包含了 pol、gag包装成分（3）包膜质粒（pMD2.G）：用其他病毒的包膜蛋白代替了 env基因.三种质粒共同转染产生不具有口我复制能力的病毒载体。

2、包装细胞：293T细胞3、菌株：大肠杆菌，用于提取质粒4、转染试剂：XTREME-GENE （-20°C保存，不可分装），一种脂质与其他组份构成的混合物5、浓缩试剂（配好后4°C保存，原材料室温保存）：5X PEG8000/NaCl溶液（聚乙二醇）：NaCl 8. 766 g; PEG8000 50g溶解在200ml Milli-Q纯水中，高圧蒸汽灭菌**也可直接从公司买來病毒液（-80°C封口膜封口冻存管保存，4°C保存3夭）：滴度一般为 108TU/ml6x 10mg/ml polybrene （-20°C分装保存）:漠化己二甲鞍。

是带正电的小分子,与细胞表面的阴离子结合，提高慢病毒对细胞的感染效率，通常加入polybrene能提高感染效率2〜10倍。

有一定细胞毒性，需要摸索浓度（l-10Ug/ml） 7、无血清培养基： optimen8、贴壁细胞（复苏后3代以上的细胞）9、puromycin：卩票吟霉索,用于筛选稳转细胞二、具体步骤病毒包装与收集（中皿，转染步骤类似于瞬转）第一天1、种板，10X105个293T细胞，加入全培养基双抗DMEM 4-5ml,过夜2、配制5XPEG8000/NaCl 溶液称取 NaCl 8. 766 g; PEG8000 50g 溶解在 200ml Milli-Q 纯水中；121 摄氏度 30min 湿热灭绝30min：保存在4°C第二夭1、配管 A 管试剂 PLKO. 1/pCDH psPAX2 pMD2. G Opti B 管 XTREME-GENE Opti 加样＞/ul 推荐量/ul、Pg合计3 1 2 200 6 200 206 206 A+B混合室温静置20min 2、加入2ml全培养基DMEM3、将1加入2,孵育10h,换成5ml全培养基1/5 （稳转）竹密岂妨流水过蛮鬼2021. 3山高哪碍野云飞第四天第五夭：1、9:00和17:00各收取一次5ml培养液，共20ml （-80°C保存）2、过滤：用孔径为0. 45mm的过滤器除去上清中的293T细胞3、加入5ml 5XPEG8000/NaCl溶液,每 30min-lh上下摇匀一次4、4°C过夜第六夭1、4°C, 3500rpm» 20min2、弃上清，倒扣纸上静置l-2min,吸干残余液体3、加入120-150 P 1 PBS,缓慢吹打，以防形成气溶胶4、50U1分装，-80°C保存。

慢病毒生产及使用操作手册

3
地址：上海市徐汇区斜土路 1175 号景泰大厦 1503 实验室：上海市张江高科技园区蔡伦路 150 号 1 号楼 2 楼邮箱：service@
400-092-0065 021-54121689
慢病毒生产及使用操作手册
当细胞生长到汇合率达到 80%~90%时需要对细胞进行传代操作，以扩大细胞数量，维持细胞良好的生长状态。
（三）做脂转 complex DMEM 需在 37 度水浴中预热，LipFiterTM 转染试剂需恢复至室温方可使用，
使用前需摇匀。
转染每瓶 T75 的 complex 成分如下：
pspax
10μg
PMD2G pHBLVTM 系列载体
10μg 10μg
转染后 6h 换新鲜培液。
注：LipoFiterTM 转染试剂为汉恒生物产品，使用说明参考 LipoFiterTM 说明书。 LipoFiterTM 转染最适的细胞密度为 50%-70%。
慢病毒生产及使用操作手册
慢病毒生产及使用操作手册
一、实验流程制备慢病毒穿梭质粒及其辅助包装原件载体质粒，三种质粒载体分别进行高
纯度无内毒素抽提，共转染 293T 细胞，转染后 6 h 更换为完全培养基，培养 48 和 72h 后，分别收集富含慢病毒颗粒的细胞上清液，病毒上清液通过超离心浓缩病毒。
2）包装质粒信息如下： PSPAX2 及 PMD2G 载体图谱和序列信息 (购自 addgene)
2、细胞株 293T，慢病毒的包装细胞，为贴壁依赖型成上皮样细胞，生长培养基为 DMEM(含 10% FBS)。贴壁细胞经培养生长增殖形成单层细胞。 3、菌株大肠杆菌菌株 DH5α。用于扩增慢病毒载体和辅助包装载体质粒。
（二）传 293T 细胞

慢病毒使用操作指南

慢使用操作指南操作指南：慢使用1、简介1.1 背景介绍1.2 慢的定义1.3 慢的应用领域2、仪器与材料2.1 实验室安全设备2.2 实验室试剂和材料2.3 慢载体和质粒3、慢的生产3.1 慢生产细胞系的选择3.2 质粒构建与筛选3.3 慢包装细胞的构建与筛选3.4 慢的生产与扩增4、慢的感染4.1 细胞培养与准备4.2 慢感染的条件优化4.3 慢感染的时间和浓度控制5、慢的转染5.1 细胞转染前的处理5.2 转染的操作条件5.3 考虑的转染效率和细胞毒性问题6、实验细胞系的维护6.1 细胞的培养和传代6.2 细胞的冻存与恢复6.3 实验细胞系的检测和验证7、实验数据记录与分析7.1 实验数据的记录和整理7.2 数据分析方法与软件使用7.3 结果的展示和解释8、安全注意事项8.1 实验操作安全措施8.2 废液处理及废弃物管理8.3 慢实验室传播的预防措施9、附件9.1 相关实验记录表格9.2 质粒和慢载体序列信息法律名词及注释：1、载体：在基因工程中，指用来携带或传递目标基因的DNA或RNA分子。

2、质粒：指自主复制的独立DNA分子，可被插入或移除目标基因，用于基因克隆、表达和操控等实验。

3、细胞培养：通过体外培养细胞的技术，提供实验所需的可控环境和条件。

4、转染：将外源DNA或RNA导入细胞内，使其表达或转录的过程。

5、传代：将细胞从一个培养器转移到另一个培养器，以维持细胞系的生长。

6、冻存：将细胞以特定的方法冷冻保存，以备将来使用。

7、废液处理：对实验过程中产生的含有有害或感染性物质的废液进行妥善处理，避免对环境和人体造成危害。

8、废弃物管理：对实验中产生的废弃物进行分类、包装和处理，符合相关法规和标准。

本文档涉及附件：1、慢生产记录表2、慢感染实验记录表3、细胞培养和传代记录表4、实验数据分析表格本文所涉及的法律名词及注释：载体、质粒、细胞培养、转染、传代、冻存、废液处理、废弃物管理。

慢病毒Cas9表达系统使用说明

慢病毒Cas9表达系统使用说明本说明书用于：¾构建Cas9蛋白表达细胞系¾在Cas9蛋白表达细胞系中敲除目的基因适用于以下产品货号货号pLV‐Cas9载体系列 CR2001，CR2002pLV‐Cas9‐Nick载体系列 CR2003，CR2004pGR载体系列 CR2011~CR2013pGR‐EGFP载体系列 CR2014~CR2016北京英茂盛业生物科技有限公司北京市昌平区沙河镇青年创业大厦B‐916Tel：010‐62495135Emai：order@Web site：目录1、产品简介 (2)1.1CRISPR/gRNA基因敲除原理 (2)1.2慢病毒Cas9表达系统特点 (2)2、Cas9表达慢病毒制备 (3)2.1试剂准备 (3)2.2简要实验流程 (4)2.3实验前准备 (4)2.4病毒制备步骤 (5)2.4 PEG纯化慢病毒 (6)3、筛选Cas9表达稳定细胞株 (7)3.1 试剂 (7)3.2 实验前准备 (7)3.3 筛选细胞系实验步骤 (8)3.4 Cas9表达细胞系检测 (9)4、用pTYNE载体对Cas9表达细胞系进行验证 (10)4.1验证Cas9蛋白表达细胞系 (10)4.2验证Cas9Nicknase蛋白表达细胞系 (11)5、pGR和pGR‐EGFP载体构建 (13)5.1 pGR和pGR‐EGFP载体图谱 (13)5.2 靶点设计 (13)5.3 pGR载体构建步骤 (14)附录1 用到的产品 (17)附录2 引物列表 (17)11、产品简介1.1CRISPR/gRNA基因敲除原理CRISPR (clustered, regularly interspaced, short palindromic repeats)是一种来自细菌降解入侵的病毒DNA或其他外源DNA的免疫机制。

在该机制中，Cas蛋白（CRISP‐associated protein）含有两个核酸酶结构域，可以分别切割两条DNA链。

吉凯基因慢病毒包装系统说明书

5．慢病毒产品优势 1）感染效率高：与传统的质粒转染相比，慢病毒感染细胞时无需转染试剂，
且转染效率高。（参见附录：图 1. 质粒转染与慢病毒感染效率比较） 2）感染谱更广：慢病毒可以高效的感染神经细胞、肝细胞、心肌细胞、内
皮细胞、干细胞、原代细胞等多种通常不容易转染的细胞。（参见附录：图 2. 慢病毒可以高效感染多种细胞）
吉凯基因慢病毒包装系统使用说明书 Lenti-Easy Packaging System
Catalog Number: LPK001（10 preps）
目录
目录.....................................................................................................................................3 1．产品组成 ............................................................................................................................. 1 2．储存条件 ............................................................................................................................. 1 3．您还需要准备的其他实验材料 ......................................................................................... 1 4．生物安全提醒 ..................................................................................................................... 2 5．慢病毒产品优势 ................................................................................................................. 2 6．慢病毒系统介绍 ................................................................................................................. 3 7．慢病毒包装流程介绍 ......................................................................................................... 4

慢病毒载体构建和包装操作手册

慢病毒载体构建及包装操作手册目录慢病毒收到后的注意事项一、整体实验流程二、实验材料三、慢病毒包装和浓缩四、感染目的细胞附1. 汉恒生物慢病毒质粒列表附2. 慢病毒滴度测定方法简介附3. 慢病毒MOI感染参数附4. 汉恒生物各病毒载体感染目的细胞比较慢病毒安全使用和注意事项➢慢病毒安全使用注意事项（*非常重要！！！*）1)慢病毒相关实验请在生物安全柜（BL-2级别）内操作。

2)操作病毒时请穿实验服，佩戴口罩和手套，尽量不要裸露双手及手臂的皮肤。

3)操作病毒时特别小心病毒溅出。

如果操作时超净工作台有病毒污染，请立即用70%乙醇加1%的SDS溶液擦拭干净。

接触过病毒的枪头，离心管，培养板，培养液请于84消毒液浸泡后统一处理。

4)如需要离心，应使用密封性好的离心管，如有必要请用封口膜封口后离心。

5)病毒相关的废弃物需要特殊收集，统一经高温灭菌处理。

6)实验完毕用香皂清洗双手。

➢慢病毒收到后的注意事项1)慢病毒的储存用户收到病毒液后在短期内即使用慢病毒进行实验，可以将病毒暂时放置于4 ℃保存（尽量一周内用完）；如需长期保存请分装后放置于-80℃。

注：a．反复冻融会降低病毒滴度（每次冻融会降低病毒滴度10%-50%）；在病毒使用过程中应尽量避免反复冻融，所以我们前期对病毒进行了分装（200 l/tube），收到后直接放置-80℃保存即可。

b．如果病毒储存时间超过6个月，我们建议在使用前重新测定病毒滴度。

2)慢病毒的稀释用户需要稀释病毒时，请将病毒取出置于冰浴融解后，使用培养目的细胞用PBS或无血清培养基(含血清或含双抗不影响病毒感染)混匀分装后置于4℃保存(请尽量一周内用完)。

一、整体实验流程二、实验材料（一）慢病毒载体、包装细胞和菌株该病毒包装系统为三质粒系统，组成为psPAX2, pMD2.G, pHBLV TM系列质粒。

1、载体信息（见附表1）2、细胞株：我们采用293T作为慢病毒的包装细胞。

该细胞系为贴壁依赖型成上皮样细胞，生长培养基为DMEM+10% FBS+双抗。

pCDH-CMV-MCS-EF1-RFP使用说明

pCDH-CMV-MCS-EF1-RFPpCDH-CMV-MCS-EF1-RFP 载体基本信息：载体名称:pCDH-CMV-MCS-EF1-RFP 质粒类型: 慢病毒表达载体；cDNA 表达载体；双启动子载体克隆方法: 多克隆位点，限制性内切酶启动子:CMV 载体大小:-- 5' 测序引物及序列:CMV-F :CGCAAATGGGCGGTAGGCGTG 3' 测序引物及序列:EF1a-R 载体标签: 无载体抗性: 氨苄青霉素（Ampicillin ）筛选标记: 嘌呤霉素克隆菌株: E.coli cells(RecA-)推荐: Stbl2 ,OmniMAX 2 T1R 宿主细胞（系）: 造血干细胞、胚胎干细胞备注: pCDH-CMV-MCS-EF1-RFP 慢病毒表达载体是基于HIV 的慢病毒载体；用于cDNA 表达和克隆；高效转染细胞，建立稳定细胞系；CMV 启动子驱动目的基因的高水平表达，EF1a 启动子驱动报告基因的中等水平的表达。

稳定性: 稳表达组成型/诱导型: 组成型病毒/非病毒: 慢病毒（HIV)pCDH-CMV-MCS-EF1-RFP 载体质粒图谱和多克隆位点信息：pCDH-CMV-MCS-EF1-RFP载体简介：This manual provides details and information necessary to generate expression constructs of your gene of interest in the pCDH cDNA Cloning and Expression Lentivectors. Specifically, it provides critical instructions on amplification and cloning cDNA into the pCDH vectors, and verification of the final expression constructs. This manual does not include information on packaging the pCDH expression constructs into pseudotyped viral particles or transducing your target cells of choice with these particles. This information is available in the user manual Lentivector Expression Systems: Guide to Packaging and Transduction of Target Cells which is available on the SBI website. Before using the reagents and material supplied with this system, please read the entire manual.基于HIV-1的pCDH 慢病毒载体特征：Multiple Cloning Site (MCS)—for cloning the gene of interest in the MCS located downstream of the CMV promoter.WPRE element—enhances stability and translation of the CMV-driven transcripts.SV40 polyadenylation signal—enables efficient termination of transcription and processing of recombinant transcripts.Hybrid RSV/5L TR promoter—provides a high level of expression of the full-length viral transcript in producer 293 cells.Genetic elements (cPPT, gag, env, L TRs)—necessary for packaging, transducing, and stablyintegrating the vira expression construct into genomic DNA.SV40 origin—for stable propagation of the pCDH plasmid in mammalian cells.pUC origin—for high copy replication and maintenance of the plasmid in E.coli cells.Ampicillin resistance gene—for selection in E.coli cells.pCDH 慢病毒表达载体的优势：Lentiviral expression vectors are the most effective vehicles for the delivery and expression of a gene of interest to almost any mammalian cell—including non-dividing cells and model organisms (C.A. Machida, 2003; M. Federico, 2003; W. C. Heiser, 2004). As with standard plasmid vectors, it is possible to introduce lentivector expression constructs in plasmid form into the cells with low-to-medium efficiency using conventional transfection protocols. However, by packaging the lentivector construct into viral particles, you can obtain highly efficient transduction of expression constructs—even with the most difficult to transfect cells, such as primary, stem, and differentiated cells. The expression construct transduced in target cells is integrated into genomic DNA and provides stable, long-term expression of the target gene.pCDH 慢病毒载体的包装载体及细胞系The expression lentivector contains the genetic elements responsible for packaging, transduction, stable integration of the viral expression construct into genomic DNA, and expression of the target gene sequence. The packaging vector provides all the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant viral particles. To produce a high titer of viral particles, expression and packaging vectors are transientlyco-transfected into producer mammalian cells (e.g., HEK 293 cells). For a detailed description of SBI’s Lentivector expression system,please refer to the Lentivector Expression System user manual.启动子的选择：SBI provides a collection of cDNA cloning and expression vectors for various applications. A gene of interest can be cloned under a CMV or EF1 promoter with or without another expression cassette for a reporter gene (copGFP or PuroR). Genes can be either expressed transiently through transfection or stably expressed in a target cell line through transduction with packaged viral particles.The major concern of cDNA expression in lentivectors is the efficiency level and stability of expression in target cell lines.The Cytomegalovirus (CMV) promoter is a strong and most commonly used viral promoter that constitutively expresses downstream genes. While the CMV promoter works perfectly in the most common cell lines, it shows poor expression in some stem cell lines and hematopoietic cell lines (R.F. Doll, 1996; E.D. Papadakis, 2004).The housekeeping elongation factor 1α (EF1) promoter has been shown to exceed and outlast CMV-mediated expression in retroviral, lentiviral, and adenoviral vectors, in hematopoietic cell lines (K. Tokushige 1997; H. Nakai, 1998; C. Teschendorf, 2002). EF1 also performs well in most common cell lines.MSCV promoter is the 5’-L TR promoter of murine stem cell virus. When a portion of the U3 regionof the 3’ HIV L TR was replaced with the U3 region of MSCV L TR, the resulted hybrid HIV/MSCVL TR has dramatically increased the transgene expression level in human CD34+ hematopoieticcells (J.K. Choi, 2001). After integration into genomic DNA, this promoter transcribes a long transcript with an intron in the 5’UTR flanked with splice donor and acce ptor sites derived fromthe lentiviral vector. Further studies found that additional CpG mutations in the MSCV L TRreduced transcriptional silencing in embryonic stem cells (C.S. Swindle, 2004). We constructed cDNA expression vectors with the CpG-deficient MSCV incorporated into the 3’ HIV L TR. After integration into genomic DNA, 3’MSCV/L TR will replace the 5’L TR and provide a high level of expression of the target gene and reporter gene downstream.SBI第三代慢病毒载体SBI offers a third generation of the most popular HIV-1 based lentivector expression systemwhich consists of three maincomponents:(1) The lentiviral expression vector (e.g., pCDH-EF1-MCS-T2A-Puro)(2) The lentiviral packaging plasmids (e.g., pPACKH1 Packaging Plasmid mix)(3) A pseudoviral particle producer cell line (e.g., 293TN cells)2A Peptide-enabled dual expression systemCoexpression of a reporter gene together with a gene of interest is a useful approach for selecting transfected or transduced cells. This is commonly achieved by using two independent internal promoters, such as CMV and EF1 in pCDH-CMV-MCSEF1- copGFP, or by linking two transgeneswith an internal ribosomal entry site (IRES) element in a single bicistronic transcript. Many dual promoter pairs have shown a high level of expression of both transgenes in standard cell lines—however, promoter interference often occurs in some cell lines. There are also two main problems that limit the use of IRES: the large size and the imbalanced expression between the first and second cistrons (H. Mizuguchi, 2000; X.Yu, 2003).The “self-cleaving” 2A peptides have been used successfully to generate multiple proteins from a single promoter in many applications (P. de Felipe, 2004; M.J. Osborn, 2005; P. de Felipe, 2006).The 2A-like sequences exist in several viruses and are used to mediate protein cleavage froma single open reading frame. Through a ribosomal skip mechanism, the 2A peptide preventsnormal peptide bond formation between the 2A glycine and the 2B proline without affecting the translation of 2B (M.L. Donnelly, 2001):SBI’s cDNA expression vectors incorporate the 2A-like sequence (T2A) from the insect virus Thosea asigna to mediate the coexpression of a reportergene with the target cDNA. Reporter genes have been cloned at either the first or second positions, and we achieved high expression levels at both locations.pCDH-CMV-MCS-EF1-RFP载体序列：pCDH-CMV-MCS-EF1-RFP其他相关慢病毒载体：Tet-pLKO-neo Tet-pLKO-puro pPACKH1-GAG pMD2.G pCMV-dR8.2-dvpr pLKO.1-GFP-shRNA pLKO.1-TRC control pLKO.1-hygro pLKO.1-TRC pCDH-MSCV-MCS-EF1-copGFP pCDH-MSCV-MCS-EF1-copGFP-T2A-Puro FUW-tetO-hOKMSFUW-tetO-hOCT4 FUW-tetO-hSOX2 FUW-tetO-hKLF4FUW pLVX-AcGFP1-N1 pLVX-AcGFP1-C1pLVX-AmCyan1-N1 pLVX-DsRed-Express2-C1 pLVX-DsRed-Express2-N1 pLVX-DsRed-Monomer-N1 pLVX-PAmCherry-C1 pLVX-PAmCherry-N1pLVX-ZsGreen1-N1 pLVX-IRES-ZsGreen1 pLVX-IRES-mCherrypLVX-mCherry-C1 pLVX-mCherry-N1 pLVX-tdTomato-C1pLKO.1-puro pLentilox 3.7 pLVX-Tet-On-Advanced pLVX-IRES-Puro pLVX-IRES-Neo pLVX-IRES-HygpLVX-EF1α-DsRed-Monomer-C1 pLVX-EF1α-AcGFP1-N1 pLVX-EF1α-AcGFP1-C1 pLVX-EF1α-mCherry-C1 pLVX-EF1α-IRES-mCherry pLVX-EF1α-IRES-ZsGreen1 pLVX-MetLuc Control pLVX-MetLuc pLVX-Hom-Mem1pLVX-Het-2 pLVX-DD-AcGFP1-Actin pPRIME-TET-GFP-FF3pSIH1-H1-CopGFP pCDH-EF1-MCS-T2A-Puro pCDH-CMV-MCS-EF1-Puro pCDF1-MCS2-EF1-copGFP pLOX-CWBmi1 pLOX-CW-CREpRSV-rev pMDLg-pRRE pLL3.7pLVX-DD-AmCyan1 Control pLVX-DD-AmCyan1 Reporter pLVX-DD-tdTomato Reporter pLVX-DD-tdTomato Control pLVX-PTuner-Green pLVX-CherryPicker2pLVX-TetOne-Puro-Luc pLVX-TetOne pLVX-TetOne-PuropLVX-TetOne-Luc pLVX-rHom-Nuc1 pLVX-rHom-Sec1pLVX-rHom-1 pLVX-Hom-Nuc1 pLVX-Het-Nuc1pLVX-PTuner pLVX-PTuner2 pLVX-DD-ZsGreen1 Reporter pLVX-Het-1 pLVX-CherryPicker Control pLVX-Tet3GpCDH-CMV-MCS-EF1-RFP-T2A-Puro pCDH-CMV-MCS-EF1-Hygro pCDH-CMV-MCS-EF1-Neo pCDH-MCS-T2A-Puro-MSCV pCDH1-MCS2-EF1-copGFP pCDF1-MCS2-EF1-Puro pCDH-EF1-MCS-T2A-copGFP pWPXL pLVX-TRE3G-ZsGreen1 pLVX-TRE3G-mCherry pLenti6.3-EmGFP-BveI miR pLenti6/V5-GW/lacZpLenti6.3/V5-GW/EmGFP pLenti6.3-MCS pLenti6.3-DsRed2-BveI miR pLenti6.3-MCS-IRES2-EGFP pLVX-shRNA2 psPAX2VSV-G pSico PGK Puro pcDNA6.2-DsRed2-MCS1 miR pcDNA6.3-EmGFP-NC- II pcDNA6.2-EmGFP-NC- I pcDNA6.2-EmGFP-BsaI miR pLenti6.3-BveI miR pLenti6.3-MCS-IRES2-DsRed2 pLEX-MCSpGIPZ pLP2 pLP1FUGW pFUGW pLOX-Ttag-iresTKpMDLg/pRRE pLentG-KOSM pCMV-dR8.91pLVX-TRE3G-Luc Control pLVX-TRE3G-IRES pCgpvpSico pSicoR pLVTHMpGensil-1 pLVX-EF1α-IRES-Puro pCDF1-MCS2-EF1-copGFP pPACKH1-REV pLVX-Het-Mem1 pLVX-shRNA1pLKO.1-puro-GFP-siRNA pPRIME-TREX-GFP-FF3 pcDNA6.2-DsRed2-BsmBI miR pCDH-MSCV-MCS-EF1-Puro pCDH-CMV-MCS-EF1-copGFP pLVX-TRE3GFUW-tetO-hMYC pLOX-TERT-iresTK pLP/VSVGFUW-M2rtTA pCDH-EF1-MCS-(PGK-Puro) pcDNA6.2-EmGFP-MCS1 miR pLVX-AmCyan1-C1 pLVX-Hom-1 pcDNA6.2-BsaI miRpLVX-DsRed-Monomer-C1 pLVX-mCherry-Actin pTRIPZpLVX-ZsGreen1-C1 pLVX-CherryPicker1 LeGO-iC2 pLVX-IRES-tdTomato pCDH-CMV-MCS-EF1-copGFP-T2A-Puro pLKO.3GpLVX-tdTomato-N1 pLVX-PTuner2-C pLVX-Puro pLVX-Tight-Puro pLVX-DD-ZsGreen1 Control pSicoR PGK Puro pLVX-EF1α-DsRed-Monomer-N1 pCDH-UbC-MCS-EF1-Hygro pLVTHpLVX-EF1α-mCherry-N1 pCDH-CMV-MCS-EF1-RFP。

慢病毒使用操作手册

慢病毒使用操作手册：1 慢病毒使用操作2 慢病毒安全使用规范3 悬浮细胞感染方法概要4 相关专业术语(详情可参考公司网站FAQ)5 细胞培养器皿的相关参数1、慢病毒使用操作手册1.1 慢病毒的储存与稀释:1.1.1 病毒的储存：用户收到病毒液后在很短时间内即使用慢病毒进行实验，可以将病毒暂时放置于4 ℃保存；如需长期保存请放置于-80℃（病毒置于冻存管，并使用封口膜封口）A．病毒可以存放于-80℃ 6个月以上；但如果病毒储存时间超过6个月，我们建议在使用前需要重新滴定病毒滴度B．反复冻融会降低病毒滴度：每次冻融会降低病毒滴度10%；因此在病毒使用过程中应仅尽量避免反复冻融，为避免反复冻融我们强烈建议客户收到病毒后按照每次的使用量进行分装。

1.1.2 病毒的稀释：用户需要稀释病毒时，请将病毒取出置于冰浴融解后，使用培养目的细胞用PBS或无血清培养基(含血清或含双抗不影响病毒感染)混匀分装后4℃保存(请尽量在三天内用完) 分装后使用。

1.2 慢病毒用于体外（In Vitro）实验：感染培养原代细胞和建系细胞1.2.1 慢病毒对各种细胞和组织的亲嗜性不同，用户使用Invabio提供的慢病毒之前可以通过查阅相关文献，了解慢病毒对您的目的细胞的亲嗜性，感染复数（MOI 值）以及在体（In Vivo）注射所需要的病毒量。

如果没有相关文献支持，可以通过感染预实验得到合适的感染复数（MOI 值）（使用24孔板检测病毒对目的细胞的亲嗜性）1.2.2 慢病毒感染目的细胞预实验1.2.2.1 慢病毒感染目的细胞预实验注意事项A．测定慢病毒对目的细胞的亲嗜性时，需要同时设置对慢病毒亲嗜性较高的细胞(HEK293T，Hela）作为平行实验的对照细胞。

B．在进行慢病毒感染实验时,可以用完全培养基（培养目的细胞用）稀释；理论上，含有血清，双抗或者其他营养因子的完全培养基不影响慢病毒的感染效率。

C． Invabio提供的病毒单位为TU/ml, 即每毫升中含有具有生物活性的病毒颗粒数。

pLenti6.3-MCS慢病毒载体使用说明

pLenti6.3-MCSpLenti6.3-MCS载体基本信息：载体名称: pLenti6.3-MCS质粒类型: 慢病毒表达载体高拷贝/低拷贝: --启动子: --克隆方法: 多克隆位点，限制性内切酶载体大小: --5' 测序引物及序列: --3' 测序引物及序列: --载体标签: --载体抗性: --筛选标记: --备注: --稳定性: --组成型: --病毒/非病毒: 慢病毒pLenti6.3-MCS载体质粒图谱和多克隆位点信息pLenti6.3-MCS载体序列pLenti6.3-MCS其他相关慢病毒载体：Tet-pLKO-neo Tet-pLKO-puro pPACKH1-GAGpMD2.G pCMV-dR8.2-dvpr pLKO.1-GFP-shRNA pLKO.1-TRC control pLKO.1-hygro pLKO.1-TRCpCDH-MSCV-MCS-EF1-copGFP pCDH-MSCV-MCS-EF1-copGFP-T2A-Puro FUW-tetO-hOKMSFUW-tetO-hOCT4 FUW-tetO-hSOX2 FUW-tetO-hKLF4FUW pLVX-AcGFP1-N1 pLVX-AcGFP1-C1pLVX-AmCyan1-N1 pLVX-DsRed-Express2-C1 pLVX-DsRed-Express2-N1 pLVX-DsRed-Monomer-N1 pLVX-PAmCherry-C1 pLVX-PAmCherry-N1 pLVX-ZsGreen1-N1 pLVX-IRES-ZsGreen1 pLVX-IRES-mCherry pLVX-mCherry-C1 pLVX-mCherry-N1 pLVX-tdTomato-C1 pLKO.1-puro pLentilox 3.7 pLVX-Tet-On-Advanced pLVX-IRES-Puro pLVX-IRES-Neo pLVX-IRES-HygpLVX-EF1α-DsRed-Monomer-C1 pLVX-EF1α-AcGFP1-N1 pLVX-EF1α-AcGFP1-C1 pLVX-EF1α-mCherry-C1 pLVX-EF1α-IRES-mCherry pLVX-EF1α-IRES-ZsGreen1 pLVX-MetLuc Control pLVX-MetLuc pLVX-Hom-Mem1pLVX-Het-2 pLVX-DD-AcGFP1-Actin pPRIME-TET-GFP-FF3pSIH1-H1-CopGFP pCDH-EF1-MCS-T2A-Puro pCDH-CMV-MCS-EF1-Puro pCDF1-MCS2-EF1-copGFP pLOX-CWBmi1 pLOX-CW-CREpRSV-rev pMDLg-pRRE pLL3.7pLVX-DD-AmCyan1 Control pLVX-DD-AmCyan1 Reporter pLVX-DD-tdTomato Reporter pLVX-DD-tdTomato Control pLVX-PTuner-Green pLVX-CherryPicker2pLVX-TetOne-Puro-Luc pLVX-TetOne pLVX-TetOne-PuropLVX-TetOne-Luc pLVX-rHom-Nuc1 pLVX-rHom-Sec1pLVX-rHom-1 pLVX-Hom-Nuc1 pLVX-Het-Nuc1pLVX-PTuner pLVX-PTuner2 pLVX-DD-ZsGreen1 Reporter pLVX-Het-1 pLVX-CherryPicker Control pLVX-Tet3GpCDH-CMV-MCS-EF1-RFP-T2A-Puro pCDH-CMV-MCS-EF1-Hygro pCDH-CMV-MCS-EF1-Neo pCDH-MCS-T2A-Puro-MSCV pCDH1-MCS2-EF1-copGFP pCDF1-MCS2-EF1-Puro pCDH-EF1-MCS-T2A-copGFP pWPXL pLVX-TRE3G-ZsGreen1 pLVX-TRE3G-mCherry pLenti6.3-EmGFP-BveI miR pLenti6/V5-GW/lacZpLenti6.3/V5-GW/EmGFP pLenti6.3-MCS pLenti6.3-DsRed2-BveI miR pLenti6.3-MCS-IRES2-EGFP pLVX-shRNA2 psPAX2VSV-G pSico PGK Puro pcDNA6.2-DsRed2-MCS1 miR pcDNA6.3-EmGFP-NC- II pcDNA6.2-EmGFP-NC- I pcDNA6.2-EmGFP-BsaI miR pLenti6.3-BveI miR pLenti6.3-MCS-IRES2-DsRed2 pLEX-MCSpGIPZ pLP2 pLP1FUGW pFUGW pLOX-Ttag-iresTKpMDLg/pRRE pLentG-KOSM pCMV-dR8.91pLVX-TRE3G-Luc Control pLVX-TRE3G-IRES pCgpvpSico pSicoR pLVTHMpGensil-1 pLVX-EF1α-IRES-Puro pCDF1-MCS2-EF1-copGFP pPACKH1-REV pLVX-Het-Mem1 pLVX-shRNA1pLKO.1-puro-GFP-siRNA pPRIME-TREX-GFP-FF3 pcDNA6.2-DsRed2-BsmBI miR pCDH-MSCV-MCS-EF1-Puro pCDH-CMV-MCS-EF1-copGFP pLVX-TRE3GFUW-tetO-hMYC pLOX-TERT-iresTK pLP/VSVGFUW-M2rtTA pCDH-EF1-MCS-(PGK-Puro) pcDNA6.2-EmGFP-MCS1 miR pLVX-AmCyan1-C1 pLVX-Hom-1 pcDNA6.2-BsaI miRpLVX-DsRed-Monomer-C1 pLVX-mCherry-Actin pTRIPZpLVX-ZsGreen1-C1 pLVX-CherryPicker1 LeGO-iC2pLVX-IRES-tdTomato pCDH-CMV-MCS-EF1-copGFP-T2A-Puro pLKO.3GpLVX-tdTomato-N1 pLVX-PTuner2-C pLVX-PuropLVX-Tight-Puro pLVX-DD-ZsGreen1 Control pSicoR PGK PuropLVX-EF1α-DsRed-Monomer-N1 pCDH-UbC-MCS-EF1-Hygro pLVTHpLVX-EF1α-mCherry-N1 pCDH-CMV-MCS-EF1-RFP。

金拓思慢病毒产品说明书

金拓思慢病毒产品说明书一、产品简介慢病毒载体是一类重组逆转录病毒载体，由于其结构和功能的特点，慢病毒载体作为一种重要的基因转移工具应用于基因治疗和细胞分子生物学研究领域。

区别于一般的逆转录病毒载体，它对分裂细胞和非分裂细胞均具有感染能力。

该载体可以将外源基因有效地整合到宿主染色体上，从而达到持久性表达。

在感染能力方面可有效地感染神经元细胞、肝细胞、心肌细胞、肿瘤细胞、内皮细胞、干细胞等多种类型细胞，达到良好的基因治疗效果。

我公司生产的重组慢病毒均以国际通用的第三代载体四质粒体系生产，通过重组改造后将含有目的基因的慢病毒骨架及其相应的作用元件组合为新质粒，并通过辅助质粒将病毒包装的元件组成重组病毒。

通过自我灭活的方式，阻止病毒自我复制。

保证了慢病毒使用过程中的良好生物安全性。

二、重要说明2.1安全操作说明1.应在Ⅱ级及以上级别生物安全柜中使用慢病毒产品。

2.虽经过改造后病毒安全性极大提高，操作中仍需佩戴口罩、手套等安全防护措施以免产生潜在危害。

3.操作中所有接触慢病毒试剂的样品、耗材、器皿等均需通过84消毒液（1:20）浸泡后，高温121℃灭活15min以上。

4.实验过程中有病毒液洒落的情况时，应用纸巾将病毒液吸干后喷洒70乙醇，并将擦干的纸巾一并高温处理以免造成其它伤害、污染环境。

2.2使用注意事项所有慢病毒产品均通过干冰低温运输，请收到产品后立即转入-80℃冰箱保存。

每次使用时提前取出病毒液放置在4℃冰箱待融化，并保存于4℃冰箱。

每次融化后请尽快使用，病毒液应尽量避免反复冻融降低病毒滴度。

三、慢病毒制备与使用3.1实验材料细胞：人贴壁细胞293T培养基：高糖DMEM培养基血清：胎牛血清抗生素：青链霉素转染试剂：Transfection-mate增强剂：Polybrene3.2病毒制备方法3.2.1细胞准备细胞复苏：1.将液氮保存细胞取出后，迅速放入37℃水浴锅内，应及时轻柔摇动加快解冻速度。

2.将完全溶解的细胞离心，1500rpm，3min。

pLKO.1.PURO 慢病毒报装Protocols

Protocols > pLKO.1 ProtocolAddgene is a global, non-profit plasmid repository dedicated to making it easier forscientists to share.pLKO.1 –TRC Cloning VectorAddgene Plasmid 10878. Protocol Version 1.0. December 2006.Copyright Addgene 2006, All Rights Reserved. This protocol is provided for yourconvenience. See “warranty information” in appendix.Table of Contents• A. pLKO.1-TRC Cloning Vector• A.1 The RNAi Consortium• A.2 Map of pLKO.1• A.3 Related plasmids• B. Designing shRNA Oligos for pLKO.1• B.1 Determine the optimal 21-mer targets in your gene• B.2 Order oligos compatible with pLKO.1• C. Cloning shRNA oligos into pLKO.1• C.1 Recommended materials• C.2 Annealing oligos• C.3 Digesting pLKO.1 TRC-Cloning Vector• C.4 Ligating and transforming into bacteria• D. Screening for Inserts• D.1 Recommended materials• D.2 Screening for inserts• E. Producing Lentiviral Particles• E.1 Recommended materials• E.2 Protocol for producing lentiviral particles• F. Infecting Target Cells• F.1 Recommended materials• F.2 Determining the optimal puromycin concentration• F.3 Protocol for lentiviral infection and selection•G. Safety•H. References•H.1 Published articles•H.2 Web resources•I. Appendix•I.1 Sequence of pLKO.1 TRC-Cloning Vector•I.2 Recipes•I.3 Warranty informationBack to TopA. pLKO.1-TRC Cloning VectorA.1 The RNAi ConsortiumThe pLKO.1 cloning vector is the backbone upon which The RNAi Consortium has built a library of shRNAs directed against 15,000 human and 15,000 mouse genes. Addgene is working with the TRC to make this shRNA cloning vector available to the scientificcommunity. Please cite Moffat et al., Cell 2006 Mar; 124(6):1283-98('PubMed”:/pubmed/16564017?dopt=abstract) in allpublications arising from the use of this vector.A.2 Map of pLKO.1pLKO.1 is a replication-incompetent lentiviral vector chosen by the TRC for expression of shRNAs. pLKO.1 can be introduced into cells via direct transfection, or can be converted into lentiviral particles for subsequent infection of a target cell line. Once introduced, the puromycin resistance marker encoded in pLKO.1 allows for convenient stable selection.Figure 1 : Map of pLKO.1 containing an shRNA insert. The original pLKO.1-TRC cloning vector has a 1.9kb stuffer that is released by digestion with AgeI and EcoRI. shRNA oligos are cloned into the AgeI and EcoRI sites in place of the stuffer. The AgeI site is destroyed in most casesA.3 Related ProductsThe following plasmids available from Addgene are recommended for use in conjunction with the pLKO.1 TRC-cloning vector.pMD2.G Envelope plasmid for producing viral particles.Note: pLKO.1 can also be used with packaging plasmid pCMV-dR8.2 dvpr and envelopeplasmid pCMV-VSVG from Robert Weinberg’s lab. For more information, visitAddgene’s Mammalian RNAi Tools page.Several other laboratories have deposited pLKO derived vectors that may also be useful for your experiment. To see these vectors, visit Addgene’s website and “search for“pLKO”“.Back to TopB. Designing shRNA Oligos for pLKO.1B.1 Determining the Optimal 21-mer Targets in your GeneSelection of suitable 21-mer targets in your gene is the first step toward efficient genesilencing. Methods for target selection are continuously being improved. Below aresuggestions for target selection.1. Use an siRNA selection tool to determine a set of top-scoring targets for your gene. Forexample, the Whitehead Institute for Biomedical Research hosts an siRNA SelectionProgram that can be accessed after a free registration(/bioc/siRNAext/). If you have MacOS X, another excellent program is iRNAi, which is provided free by the company Mekentosj (/irnai/).A summary of guidelines for designing siRNAs with effective gene silencing is includedhere:•Starting at 25nt downstream of the start codon (ATG), search for 21nt sequences that match the pattern AA(N 19 ). If no suitable match is found, search for NAR(N 17 )YNN, where N isany nucleotide, R is a purine (A,G), and Y is a pyrimidine (C,U).•G-C content should be 36-52%.•Sense 3’ end should have low stability – at least one A or T between position 15-19.•Avoid targeting introns.•Avoid stretches of 4 or more nucleotide repeats, especially repeated Ts because polyT is a termination signal for RNA polymerase III.2. To minimize degradation of off-target mRNAs, use NCBI’s BLAST program. Selectsequences that have at least 3 nucleotide mismatches to all unrelated genes.Addgene recommends that you select multiple target sequences for each gene.Some sequences will be more effective than others. In addition, demonstrating that twodifferent shRNAs that target the same gene can produce the same phenotype willalleviate concerns about off-target effects.B.2 Ordering Oligos Compatible with pLKO.1To generate oligos for cloning into pLKO.1, insert your sense and antisense sequences from step B.1 into the oligos below. Do not change the ends; these bases are important for cloning the oligos into the pLKO.1 TRC-cloning vector.Forward oligo:5’ CCGG—21bp sense—CTCGAG—21bp antisense—TTTTTG3’Reverse oligo:5’ AATTCAAAAA—21bp sense—CTCGAG—21bp antisense 3’For example, if the target sequence is (AA)TGCCTACGTTAAGCTATAC, the oligos would be:Forward oligo:5’CCGG AATGCCTACGTTAAGCTATAC CTCGAG GTATAGCTTAACGTAGGCATT TTTTTG 3’Reverse oligo:5’AATTCAAAAA AATGCCTACGTTAAGCTATAC CTCGAG GTATAGCTTAACGTAGGCATT 3’Back to TopC. Cloning Oligos into pLKO.1The pLKO.1-TRC cloning vector contains a 1.9kb stuffer that is released upon digestion with EcoRI and AgeI.The oligos from section B contain the shRNA sequence flanked by sequences that are compatible with the sticky ends of EcoRI and AgeI. Forward and reverse oligos are annealed and ligated into the pLKO.1 vector, producing a final plasmid that expresses the shRNA of interest.C.1 Recommended Materials5 μL Forward oligo5 μL Reverse oligo5 μL 10x NEB buffer 235 μL ddH2O2. Incubate for 4 minutes at 95°C in a PCR machine or in a beaker of boiling water.3. If using a PCR machine, incubate the sample at 70°C for 10 minutes then slowly cool to room temperature over the period of several hours. If using a beaker of water, remove the beaker from the flame, and allow the water to cool to room temperature. This will take a few hours, but it is important for the cooling to occur slowly for the oligos to anneal.C.3 Digesting pLKO.1 TRC Cloning Vector1. Digest pLKO.1 TRC-cloning vector with AgeI. Mix:6 μg pLKO.1 TRC-cloning vector (maxiprep or miniprep DNA)5 μL 10x NEB buffer 11 μL AgeIto 50 μL ddH2O> Incubate at 37°C for 2 hours.2. Purify with Qiaquick gel extraction kit. Elute in 30 μL of ddH2O.3. Digest eluate with EcoRI. Mix:30 μL pLKO.1 TRC-cloning vector digested with AgeI5 μL 10x NEB buffer for EcoRI1 μL EcoRI14 μL ddH2O> Incubate at 37°C for 2 hours.4. Run digested DNA on 0.8% low melting point agarose gel until you can distinctly see 2 bands, one 7kb and one 1.9kb. Cut out the 7kb band and place in a sterile microcentrifuge tube.When visualizing DNA fragments to be used for ligation, use only long-wavelength UV light. Short wavelength UV light will increase the chance of damaging the DNA.5. Purify the DNA using a Qiaquick gel extraction kit. Elute in 30 μL of ddH2O.6. Measure the DNA concentration.C.4 Ligating and Transforming into Bacteria1. Use your ligation method of choice. For a standard T4 ligation, mix:2 μL annealed oligo from step C.2.20 ng digested pLKO.1 TRC-cloning vector from step C.3. (If you were unable to measure the DNA concentration, use 1 μL)2 μL 10x NEB T4 DNA ligase buffer1 μL NEB T4 DNA ligaseto 20 μL ddH2O> Incubate at 16°C for 4-20 hours.2. Transform 2 μL of ligation mix into 25 μL competent DH5 alpha cells, following manufacturer’s protocol. Plate on LB agar plates containing 100 μg/mL ampicillin or carbenicillin (an ampicillin analog).Back to TopD. Screening for InsertsYou may screen for plasmids that were successfully ligated by restriction enzyme digestion. However, once you have identified the positive clones, it is important to verify the insert by conducting a sequencing reaction.D.1 Recommended MaterialsDay 1:1. Innoculate 5 colonies from each ligation into LB + 100 μg/mL ampicillin or carbenicillin.Day 2:2. Spin down the cultures and use a miniprep kit to obtain DNA.3. Conduct a restriction digest with EcoRI and NcoI:• 1 μg miniprep DNA• 2 μL 10x NEB buffer for EcoRI•0.8 μL EcoRI•0.8 μL NcoI•to 20 μL ddH2O> Incubate at 37°C for 1-2 hours.4. Run the digestion products on a 1% agarose gel. You should see two fragments, a 2kb fragment and a 5kb fragment.5. Sequence positive clones with pLKO.1 sequencing primer(5’CAA GGC TGT TAG AGA GAT AAT TGG A 3’).You may need to adjust the sequencing conditions if the DNA polymerase has difficulty reading through the secondary structure of the hairpin sequence.Back to TopE. Producing Lentiviral ParticlesBefore this step, you must contact your institution’s Bio-Safety office to receive permission and institution-specific instructions. You must follow safety procedures and work in an environment (e.g. BL2+) suitable for handling HIV-derivative viruses.For transient knockdown of protein expression, you may transfect plasmid DNA directly into the target cells. The shRNA will be expressed, but the DNA is unlikely to be integrated into the host genome.For stable loss-of-function experiments, Addgene recommends that you generate lentiviral particles and infect the target cells. Addition of puromycin will allow you to select for cells that stably express your shRNA of interest.E.1 Recommended Materialsc. In polypropylene microfuge tubes (do NOT use polystyrene tubes), make a cocktail for each transfection:• 1 μg pLKO.1 shRNA plasmid•750 ng psPAX2 packaging plasmid•250 ng pMD2.G envelope plasmid•to 20 μl serum-free OPTI-MEMYou may want to vary the ratio of shRNA plasmid, packaging plasmid, and envelope plasmid to obtain the ratio that gives you the optimal viral production.d. Create a master mix of FuGENE® 6 transfection reagent in serum-free OPTI-MEM. Calculate the amount of Fugene® and OPTI-MEM necessary given that each reaction will require 6 μL FuGENE® + 74 μL OPTI-MEM. For example:•1x master mix: 6 μL FuGENE® + 74 μL OPTI-MEM•5x master mix: 30 μL FuGENE® + 370 μL OPTI-MEM•10x master mix: 60 μL FuGENE® + 740 μL OPTI-MEMIn a polypropylene tube, add OPTI-MEM first. Pipette FuGENE® directly intothe OPTI-MEM– do not allow FuGENE® to come in contact with the walls of thetube before it has been diluted. Mix by swirling or gently flicking the tube. Incubate for 5 minutes at room temperature.e. Add 80 μL of FuGENE® master mix to each tube from step c for a total volume of 100 μL. Pipette master mix directly into the liquid and not onto the walls of the tube. Mix by swirling or gently flicking the tube.f. Incubate for 20-30 minutes at room temperature.g. Retrieve HEK-293T cells from incubator. The cells should be 50-80% confluent and in DMEM that does not contain antibiotics.h. Without touching the sides of the dish, gently add DNA:FuGENE® mix dropwise to cells. Swirl to disperse mixture evenly. Do not pipette or swirl too vigorously, as you do not want to dislodge the cells from the plate.i. Incubate cells at 37°C, 5% CO2 for 12-15 hours.Day 3:j. In the morning, change the media to remove the transfection reagent. Replace with 5 mL fresh DMEM + 10% FBS + penicillin/streptomycin. Pipette the media onto the side of the plate so as not to disturb the transfected cells.k. Incubate cells at 37°C, 5% CO2 for 24 hours.Day 4:l. Harvest media from cells and transfer to a polypropylene storage tube. The media contains your lentiviral particles. Store at 4°C.m. Add 5 mL of fresh media containing antibiotics to the cells and incubate at 37°C, 5% CO2 for 24 hours.Day 5:n. Harvest media from cells and pool with media from Day 4. Spin media at 1,250 rpm for 5 minutes to pellet any HEK-293T cells that were inadvertently collected during harvesting.In lieu of centrifugation, you may filter the media through a 0.45 μm filter to remove the cells. Do not use a 0.2 μm filter, as this is likely to shear the envelope of your virus.o. Virus may be stored at 4°C for a few days, but should be frozen at -20°C or -80°C for long-term storage.Freeze/thaw cycles decrease the efficiency of the virus, so Addgene recommends that you use the virus immediately or aliquot the media into smaller tubes to prevent multiple freeze/thaw cycles.Back to TopF. Infecting Target CellsLentiviral particles can efficiently infect a broad range of cell types, including both dividing and non-dividing cells. Addition of puromycin will allow you to select for cells that are stably expressing your shRNA of interest.F.1. Recommended MaterialsDay 2:b. The target cells should be approximately 80-90% confluent.c. Dilute puromycin in the preferred culture media for your target cells. The final concentration of puromycin should be from 1-10 μg/mL in 1 μg/mL increments.d. Label plates from 1-10 and add appropriate puromycin-containing media to cells.Days 3+:e. Examine cells each day and change to fresh puromycin-containing media every other day.f. The minimum concentration of puromycin that results in complete cell death after 3-5 days is the concentration that should be used for selection in your experiments. (You may wish to repeat this titration with finer increments of puromycin to determine a more precise optimal puromycin concentration.)F.3. Protocol for Lentiviral Infection and SelectionDay 1:a. Plate target cells and incubate at 37°C, 5% CO2 overnight.Day 2:b. Target cells should be approximately 70% confluent. Change to fresh culture media containing 8 μg/mL polybrene.Polybrene increases the efficiency of viral infection. However, polybrene is toxic to some cell lines. In these cell lines, substitute protamine sulfate for polybrene.c. Add lentiviral particle solution from step E. For a 6 cm target plate, add between0.05-1 mL v irus (add ≥0.5 mL for a high MOI, and ≤0.1 mL for a low MOI). Scale the amount of virus added depending on the size of your target plate.MOI (multiplicity of infection) refers to the number of infecting viral particles per cell. Addgene recommends that you test a range of MOIs to determine the optimal MOI for infection and gene silencing in your target cell line.d. Incubate cells at 37°C, 5% CO2 overnight.Day 3:e. Change to fresh media 24 hours after infection.If viral toxicity is observed in your cell line, you may decrease the infection time to between 4 – 20 hours. Remove the virus-containing media and replace with fresh media. Do not add puromycin until at least 24 hours after infection to allow for sufficient expression of the puromycin resistance gene.f. To select for infected cells, add puromycin to the media at the concentration determined in step E.2.Addgene recommends that you maintain one uninfected plate of cells in parallel. This plate will serve as a positive control for the puromycin selection.Days 4+:g. Change to fresh puromycin-containing media as needed every few days.h. Assay infected cells. The following recommendations are guidelines for the number of days you should wait until harvesting your cells. However, you should optimize the time based on your cell line and assay:Assay Days post-infectionmRNA knockdown≥ 3 days(quantitative PCR)Protein knockdown (western blot) ≥ 4 daysPhenotypic assay ≥ 4 daysBack to TopG. SafetyBL2 safety practices should be followed when preparing and handling lentiviral particles. Personal protective clothing should be worn at all times. Use plastic pipettes in place of glass pipettes or needles. Liquid waste should be decontaminated with at least 10% bleach. Laboratory materials that come in contact with viral particles should be treated as biohazardous waste and autoclaved. Please follow all safety guidelines from your institution and from the CDC and NIH for work in a BL2 facility.If you have any questions about what safety practice to follow, please contact your institution’s safety office.To obtain the MSDS for this product, visit /sitemap and followthe MSDS link.Back to TopH. ReferencesH.1. Published ArticlesKhvorova A et. al. 2003. Functional siRNAs and miRNAs exhibit strand bias. Cell 115:209-216. (PubMed)Moffat J et. al. 2006. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124:1283-1298. (PubMed)Naldini L et. al. 1996. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272:263-267. (PubMed)Schwarz DS et. al. 2003. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115:199-208. (PubMed)Stewart SA et. al. 2003. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA 9(4):493-501. (PubMed)Zufferey R et. al. 1997. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 15(9):871-5. (PubMed)Zufferey R et. al. 1998. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 72(12):9873-80. (PubMed)H.2. Web resourcesAddgene’s mammalian RNAi website:/mammalianrnaiThe RNAi Consortium (TRC): /genome_bio/trc/rnai.htmlBackground on RNAimechanism: /focus/rnai/animations/animation/animation.htmWhitehead siRNA Selection Program: /bioc/siRNAext/Mekentosj iRNAi Program: /irnai/Back to TopI. AppendixI.1. Sequence of pLKO.1 TRC-Cloning VectorClick here to see the sequence of pLKO.1 TRC-cloning vector. The vector is 8901 base pairs total, and the stuffer insert is shown in all capital letters.I.2. RecipesLuria Broth Agar (LB agar) + antibioticPer 40 grams of powder from American Bioanalytical catalog # AB01200-02000, LB contains:10g tryptone5g yeast extract10g sodium chloride15g agar> Prepare LB agar solution by dissolving 40g of LB powder in 1L of distilled water. Autoclave and cool to 55°C. Add 1mL of 100mg/mL ampicillin or carbenicillin to obtain a final concentration of 100 μg/mL antibiotic. Pour plates and store at 4°C.Hexadimethrine Bromide (Polybrene)Prepare a 1mg/mL solution of polybrene (Sigma-Aldrich catalog #H9268) in 0.9% NaCl. Autoclave to sterilize. Stock solution is stable at 4°C for up to one year. The powder form of polybrene is stable at 4°C for several years.Protamine SulfateStore protamine sulfate (MP Biomedicals catalog #194729) at 4°C. Freely soluble in hot water and slightly soluble in cold water.PuromycinPrepare a 50mg/mL stock solution of puromycin (Sigma-Aldrich catalog #P8833) in distilled water. Sterilize by passing through a 0.22 μm filter. Store aliquots at -20°C.I.3. Warranty InformationAddgene is committed to providing scientists with high-quality goods and services. Addgene makes every effort to ensure the accuracy of its literature, but realizes that typographical or other errors may occur. Addgene makes no warranty of any kind regarding the contents of any literature. Literature are provided to you as a guide and on an “AS IS” “AS AVAILABLE” basis without warranty of any kind either expressed or implied, including but not limited to the implied warranties of fitness for a particular purpose, non-infringement, typicality, safety and accuracy.The distribution of any literature by Addgene is not meant to carry with it, and does not grant any license or rights of access or use to the materials described in the literature.The distribution of materials by Addgene is not meant to carry with it, and does not grant any license, express or implied, under any patent. All transfers of materials from Addgene to any party are governed by Addgene’s Terms of Use, Addgene’s Terms of Purchase, and applicable Material Transfer Agreements between the party that deposited the material at Addgene and the party receiving the material.。

慢病毒包装试剂盒说明书

YRGene 慢病毒包装试剂盒说明书产品编号：LPK010 产品规格：10个10cm 皿产品简介：赢润生物的慢病毒包装试剂盒包括如下成分：（1）优化配比的慢病毒包装辅助质粒混合物，可兼容大多数慢病毒表达载体。

（2）高效转染试剂（Invitrogen Lip2000原装产品分装，293T 细胞转染效率接近 100%）。

（3 ）高效率的慢病毒浓缩液，无需超速离心，也不需要价格昂贵的过滤柱，快速富集病毒粒子，其优势在于操作安全简单，对设备要求低，产毒效率高，能够快速、高效地收获高滴度病毒。

产品组成：1. 转染前，传代 293FT 细胞于10cm 培养皿中（例如，接种 1 x 107细胞于10cm 培养皿中，使用完全培养基DMEM+10%FBS培养），当细胞密度能够达到 90-95%即可进行转染。

Tips :培养基里面不要添加抗生素。

2. 转染前1-3小时，更换培养基，加入7ml 新鲜的完全培养基（DMEM+10%FBS ）,注意不要添加抗生素。

3. 准备转染。

在5ml 离心管中，分别配制 A 管与B 管试剂（Tube A and Tube B ）配好后，放置5min ，然后将A 管缓慢加入B 管，混合均匀。

室温放置20min ，使得脂质体-DNA 混合物形成。

Tips :混合后可能会出现淡淡的乳白状，不会影响转染。

然后将混合液逐滴均匀加入 10cm 培养皿，轻微混匀。

置于37C, 5% CO ?培养箱中培养过夜。

4. 第三天，更换培养基，加入 10ml 的完全培养基，同样注意不要加抗生素。

5. 转染后48-72h 后收取上清，转移至 15ml 离心管。

Tips :上清里面含有病毒，请小心操作。

6. 3000rpm 在4C 离心15min ，去除沉淀。

7. 上清液用0.45卩m 滤器过滤后转移到新的离心管中。

慢病毒浓缩：1. 每10ml 过滤后的病毒初始液，加入 Concen Solution 3ml ，每20-30min 混合一次，共进行 3-5次。

pLenti6.3-MCS-IRES2-EGFP慢病毒载体使用说明

pLenti6.3-MCS-IRES2-EGFPpLenti6.3-MCS-IRES2-EGFP载体基本信息：载体名称: pLenti6.3-MCS-IRES2-EGFP质粒类型: 慢病毒表达载体高拷贝/低拷贝: --启动子: --克隆方法: 多克隆位点，限制性内切酶载体大小: --5' 测序引物及序列: --3' 测序引物及序列: --载体标签: EGFP载体抗性: --筛选标记: --备注: --稳定性: --组成型: --病毒/非病毒: 慢病毒pLenti6.3-MCS-IRES2-EGFP载体质粒图谱和多克隆位点信息：pLenti6.3-MCS-IRES2-EGFP载体序列pLenti6.3-MCS-IRES2-EGFP其他相关慢病毒载体：Tet-pLKO-neo Tet-pLKO-puro pPACKH1-GAGpMD2.G pCMV-dR8.2-dvpr pLKO.1-GFP-shRNA pLKO.1-TRC control pLKO.1-hygro pLKO.1-TRCpCDH-MSCV-MCS-EF1-copGFP pCDH-MSCV-MCS-EF1-copGFP-T2A-Puro FUW-tetO-hOKMSFUW-tetO-hOCT4 FUW-tetO-hSOX2 FUW-tetO-hKLF4FUW pLVX-AcGFP1-N1 pLVX-AcGFP1-C1pLVX-AmCyan1-N1 pLVX-DsRed-Express2-C1 pLVX-DsRed-Express2-N1 pLVX-DsRed-Monomer-N1 pLVX-PAmCherry-C1 pLVX-PAmCherry-N1 pLVX-ZsGreen1-N1 pLVX-IRES-ZsGreen1 pLVX-IRES-mCherry pLVX-mCherry-C1 pLVX-mCherry-N1 pLVX-tdTomato-C1 pLKO.1-puro pLentilox 3.7 pLVX-Tet-On-Advanced pLVX-IRES-Puro pLVX-IRES-Neo pLVX-IRES-HygpLVX-EF1α-DsRed-Monomer-C1 pLVX-EF1α-AcGFP1-N1 pLVX-EF1α-AcGFP1-C1 pLVX-EF1α-mCherry-C1 pLVX-EF1α-IRES-mCherry pLVX-EF1α-IRES-ZsGreen1 pLVX-MetLuc Control pLVX-MetLuc pLVX-Hom-Mem1pLVX-Het-2 pLVX-DD-AcGFP1-Actin pPRIME-TET-GFP-FF3pSIH1-H1-CopGFP pCDH-EF1-MCS-T2A-Puro pCDH-CMV-MCS-EF1-Puro pCDF1-MCS2-EF1-copGFP pLOX-CWBmi1 pLOX-CW-CREpRSV-rev pMDLg-pRRE pLL3.7pLVX-DD-AmCyan1 Control pLVX-DD-AmCyan1 Reporter pLVX-DD-tdTomato Reporter pLVX-DD-tdTomato Control pLVX-PTuner-Green pLVX-CherryPicker2pLVX-TetOne-Puro-Luc pLVX-TetOne pLVX-TetOne-PuropLVX-TetOne-Luc pLVX-rHom-Nuc1 pLVX-rHom-Sec1pLVX-rHom-1 pLVX-Hom-Nuc1 pLVX-Het-Nuc1pLVX-PTuner pLVX-PTuner2 pLVX-DD-ZsGreen1 Reporter pLVX-Het-1 pLVX-CherryPicker Control pLVX-Tet3GpCDH-CMV-MCS-EF1-RFP-T2A-Puro pCDH-CMV-MCS-EF1-Hygro pCDH-CMV-MCS-EF1-Neo pCDH-MCS-T2A-Puro-MSCV pCDH1-MCS2-EF1-copGFP pCDF1-MCS2-EF1-Puro pCDH-EF1-MCS-T2A-copGFP pWPXL pLVX-TRE3G-ZsGreen1 pLVX-TRE3G-mCherry pLenti6.3-EmGFP-BveI miR pLenti6/V5-GW/lacZpLenti6.3/V5-GW/EmGFP pLenti6.3-MCS pLenti6.3-DsRed2-BveI miR pLenti6.3-MCS-IRES2-EGFP pLVX-shRNA2 psPAX2VSV-G pSico PGK Puro pcDNA6.2-DsRed2-MCS1 miR pcDNA6.3-EmGFP-NC- II pcDNA6.2-EmGFP-NC- I pcDNA6.2-EmGFP-BsaI miR pLenti6.3-BveI miR pLenti6.3-MCS-IRES2-DsRed2 pLEX-MCSpGIPZ pLP2 pLP1FUGW pFUGW pLOX-Ttag-iresTKpMDLg/pRRE pLentG-KOSM pCMV-dR8.91pLVX-TRE3G-Luc Control pLVX-TRE3G-IRES pCgpvpSico pSicoR pLVTHMpGensil-1 pLVX-EF1α-IRES-Puro pCDF1-MCS2-EF1-copGFP pPACKH1-REV pLVX-Het-Mem1 pLVX-shRNA1pLKO.1-puro-GFP-siRNA pPRIME-TREX-GFP-FF3 pcDNA6.2-DsRed2-BsmBI miR pCDH-MSCV-MCS-EF1-Puro pCDH-CMV-MCS-EF1-copGFP pLVX-TRE3GFUW-tetO-hMYC pLOX-TERT-iresTK pLP/VSVGFUW-M2rtTA pCDH-EF1-MCS-(PGK-Puro) pcDNA6.2-EmGFP-MCS1 miR pLVX-AmCyan1-C1 pLVX-Hom-1 pcDNA6.2-BsaI miRpLVX-DsRed-Monomer-C1 pLVX-mCherry-Actin pTRIPZpLVX-ZsGreen1-C1 pLVX-CherryPicker1 LeGO-iC2pLVX-IRES-tdTomato pCDH-CMV-MCS-EF1-copGFP-T2A-Puro pLKO.3GpLVX-tdTomato-N1 pLVX-PTuner2-C pLVX-PuropLVX-Tight-Puro pLVX-DD-ZsGreen1 Control pSicoR PGK PuropLVX-EF1α-DsRed-Monomer-N1 pCDH-UbC-MCS-EF1-Hygro pLVTHpLVX-EF1α-mCherry-N1 pCDH-CMV-MCS-EF1-RFP。

慢病毒操作资料说明

慢病毒操作资料说明慢病毒操作资料说明1、Hexadimethrine bromide.pdf：这是Hexadimethrine bromide（别名Polybrene）的说明书，该试剂用于提高慢病毒的感染效率，在病毒滴度测定和病毒感染实验中均需使用；2、Lenti-X™ Concentrator.pdf：这是Lenti-X Concentrator的说明书，该试剂用于病毒粒子的浓缩纯化；3、Lenti-X™ Lentiviral Expression Systems User Manual.pdf：这是过表达慢病毒（用于基因过表达）的操作说明书4、Lenti-X™ shRNA Expression Systems User Manual.pdf：这是干扰慢病毒（用于基因干扰）的操作说明书5、pLVX-shRNA1 Vector Information.pdf：pLVX-shRNA1慢病毒干扰载体说明书6、pLVX-IRES-Neo Vector Information.pdf：pLVX-IRES-Neo 慢病毒过表达载体说明书7、慢病毒（Lentivirus）载体.pdf：慢病毒载体介绍8、Lenti-X™ Lentiviral Packaging Systems FAQs.pdf：慢病包装系统常见问题介绍9、病毒纯化-PEG6000.doc：病毒纯化方法10、病毒滴度测定-针对没有绿色荧光蛋白标记的病毒.doc：病毒滴度测定-针对没有绿色荧光蛋白标记的病毒；11、病毒滴度测定-针对有绿色荧光蛋白标记的病毒.doc：病毒滴度测定-针对有绿色荧光蛋白标记的病毒；12、Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors.pdf：慢病毒包装、纯化、滴度测定操作指南，供理论学习；13、C0508磷酸钙法细胞转染试剂盒.pdf：磷酸钙转染方法；14、慢病毒实验方法.doc：慢病毒包装报告单；15、慢病毒包装、纯化、滴度测定及感染.doc：慢病毒包装、纯化、滴度测定及感染操作指南；。

慢病毒使用操作手册

慢病毒使用操作手册简介：慢病毒（Lentivirus）是一种常用于生命科学研究中的病毒载体，其具有较强的基因转染能力和稳定的基因表达特性。

本操作手册旨在向研究者提供一个详细的慢病毒使用指南，帮助他们顺利进行慢病毒基因转染实验并获得准确可靠的研究结果。

一、慢病毒基本原理慢病毒属于反转录病毒，其基因组为单链RNA。

慢病毒在寄主细胞内通过逆转录过程将RNA转录为DNA，随后将DNA插入宿主基因组中。

这使得慢病毒成为将外源基因稳定集成到宿主基因组中的理想工具。

二、慢病毒使用前准备1. 实验室条件准备：确保工作台面干净整洁，并准备好所需的培养物、培养器具和试剂。

2. 慢病毒载体制备：根据实验需要，选择合适的慢病毒载体，并通过慢病毒包装系统将目标基因插入载体中。

三、慢病毒转染实验步骤1. 细胞培养：将目标细胞接种在培养皿中，并选择合适的培养基进行细胞培养。

2. 慢病毒感染：将预制的慢病毒悬液加入培养皿中，控制感染浓度和时间，以实现最佳的感染效果。

3. 筛选标记：根据实验需要，在感染后适当的时间点添加筛选标记物，如抗生素或荧光标记剂。

4. 选择和扩增：将受筛选标记影响的细胞单克隆分离，扩增和保存。

5. 验证表达：使用合适的实验方法，如western blot或PCR等，来确认目标基因的表达情况。

6. 结果分析：对实验结果进行统计学分析，并绘制适当的图表。

四、注意事项和常见问题解决方案1. 实验前应认真阅读文献，了解慢病毒的基本原理和实验操作流程。

2. 制备慢病毒载体时，应仔细验证目标基因的序列和正确插入。

3. 慢病毒感染时应注意控制感染浓度和时间，避免细胞毒性和非特异性感染。

4. 筛选标记物的选择应根据实验需要和细胞类型进行合理选择。

5. 实验过程中，注意严格遵守实验室安全和生物安全操作规范。

6. 常见问题解决方案：如遇到感染效率低或细胞毒性问题，可以尝试优化感染条件或调整细胞培养条件。

如果基因表达不稳定，可以尝试选择合适的筛选标记物或优化基因载体。

慢病毒(过表达)包装步骤

慢病毒（过表达）包装步骤秦超1.转染复苏293T细胞，传2-3代进行转染，转染推荐使用合元公司的慢病毒转染试剂。

转染步骤：（以10cm培养皿为例）⑴最好在铺细胞后20h左右进行转染，控制转染前细胞密度70%-90%，保证细胞处于良好的状态，转染前一小时把一半培养基（约5ml）换成新的（含血清，因为此转染试剂不需换液）。

⑵加psin 10ug，pspax2 10ug，pmd2.g 5ug于800ul opti-mem，混匀⑶加40ul慢病毒转染试剂于800ul opti-mem，混匀，室温静置5min⑷将⑶所得的转染试剂稀释液滴加到⑵所得到的质粒稀释液中，边加边轻轻混匀，室温放置20min⑸取出细胞培养皿，将⑷得到的质粒转染试剂复合体加入到细胞培养基中，前后轻轻推摇使混合均匀，放回培养箱。

2.收毒(36-48h)收毒前如果质粒带有荧光标签可先看一下转染效率，一般达到60%即可。

⑴将培养皿中的病毒上清液吸出到15cm离心管中，然后2000rpm离心10min，以沉淀细胞碎片。

⑵取上清用0.22um滤清过滤到浓缩管（用蛋白质浓缩管即可）中。

4000rpm离心至所需体积。

⑶浓缩完毕后，吸出浓缩后的病毒液，按每次的接毒量分装，-80℃冻存。

由于反复冻融会降低慢病毒滴度，因此避免反复冻融。

3.接毒接毒前12-20h铺细胞，使接毒时细胞密度约为40%-50%，务必使用生长状态良好的细胞。

将分装好的慢病毒滴加到细胞中，加polybrene使其终浓度为8ug/ml细胞密度60%-70%时可以再接毒一次。

4.检测及培养细胞系(48h)如果带有荧光标签可直接显微镜看一下感染效率，如需用药杀用puromycin杀三天（对照组完全杀死），剩下的即为基因整合进去的细胞。

如需培养成细胞系，可继续培养。

如果剩下的细胞较少可用高浓度血清，待细胞聚团时用胰酶消化一下，使细胞铺匀。