pLOX-Ttag-iresTK慢病毒载体使用说明
慢病毒载体使用手册
LentiCRISPRv2 and lentiGuide-Puro: lentiviral CRISPR/Cas9 and single guide RNA CRISPR (C lustered R egularly I nterspaced S hort P alindromic R epeats) is a microbial nuclease system involved in defense against invading phages and plasmids. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage. Lentiviral CRISPR/Cas can infect a broad variety of mammalian cells by co-expressing a mammalian codon-optimized Cas9 nuclease along with a single guide RNA (sgRNA) to facilitate genome editing (Shalem*, Sanjana*, et al., Science 2014). Protocols for cloning into the lentiviral transfer plasmid and general considerations for producing lentivirus are described below. Separate protocols are available for amplifying the genome-scale CRISPR knock-out (GeCKO) libraries. This protocol is for creating individual lentiviral CRISPR plasmids targeting a single genomic locus.lentiCRISPRv2 (one vector system): This plasmid contains two expression cassettes, hSpCas9 and the chimeric guide RNA. The vector can be digested using BsmB I, and a pair of annealed oligos can be cloned into the single guide RNA scaffold. The oligos are designed based on the target site sequence (20bp) and needs to be flanked on the 3' end by a 3bp NGG PAM sequence, as shown on the next page.lentiGuide-Puro (two vector system): This plasmid expressed only the chimeric guide RNA. It does not contain Cas9. Please use lentiCas9-Blast (a separate lentiviral construct that delivers hSpCas9 and blasticidin resistance) to first integrate Cas9 into your cell line. The lentiGuide-Puro vector can be digested using BsmB I, and a pair of annealed oligos can be cloned into the single guide RNA scaffold. The oligos are designed based on the target site sequence (20bp) and needs to be flanked on the 3' end by a 3bp NGG PAM sequence, as shown on the next page.Which vector to use: lentiCRISPRv2 is identical to the original lentiCRISPRv1 but produces nearly 10X higher titer virus. lentiGuide-Puro produces >100X higher titer virus over lentiCRISPRv1 and should be used in cell lines where Cas9 has already been integrated in (e.g. using the separate lentiCas9-Blast lentivirus). For applications where Cas9 cannot first be introduced (e.g. primary cells), lentiCRISPRv2 is recommended. After transduction, use puromycin to select for cells with lentiCRISPRv2 or lentiGuide-Puro.Lentiviral production: Before starting any lentiviral work, please ensure compliance with your Environmental Health and Safety office and government/organization/university. Briefly, to make lentivirus, a transfer plasmid (e.g. lentiCRISPRv2 or lentiGuide-Puro) must be co-transfected into HEK293(F)T cells with the packaging plasmids pVSVg (AddGene 8454) and psPAX2 (AddGene 12260). As a positive control for viral production, we often use a CMV-EGFP lentiviral transfer plasmid (eg. AddGene 19319).Target design notes and online resources: For application of Cas9 for site-specific genome editing in eukaryotic cells and organisms, we have computationally identified suitable target sites for the S. pyogenes Cas9 and calculated most likely off-targets within the genome. Please visit to access these Cas9 target design tools. Complete plasmid sequences, protocols, a discussion forum and additional information can be found at the Zhang Lab GeCKO website: /gecko/ . Citation: Please reference the following publications for the use of this material.Improved lentiviral vectors and genome-wide libraries for CRISPR screening. Sanjana NE*, Shalem O*, Zhang F. Nature Methods (2014).Genome-scale CRISPR-Cas9 knockout screening in human cells. Shalem O*, Sanjana NE*, Hartenian E, Shi X, Scott DA, Mikkelsen T, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F (2014). Science, 343, 83-7. DOI: 10.1126/science.1247005Target Guide Sequence Cloning ProtocolIn order to clone the target sequence into the lentiCRISPRv2 or lentiGuide-Puro backbone, synthesize two oligos of the following form. All plasmids have the same overhangs after BsmBI digestion and the same oligos can be used for cloning into lentiCRISPRv2, lentiGuide-Puro or lentiCRISPRv1.Example oligo design : Note that the NGG PAM is not included in the designed oligos. Oligonucleotide ordering tips : Standard de-salted oligos (usually the most inexpensive synthesis) are sufficient forcloning. If not already resuspended, dilute each oligo to 100 µM in sterile water or TE.* * * * *Lentiviral vector digestion, oligo annealing and cloning into digested vector:1. Digest and dephosphorylate 5ug of the lentiviral CRISPR plasmid with BsmB I for 30 min at 37C: 5 ug lentiCRISPRv2 or lentiGuide-Puro 3 ul FastDigest BsmB I (Fermentas) 3 ul FastAP (Fermentas) 6 ul 10X FastDigest Buffer 0.6 ul 100 mM DTT (freshly prepared) X ul ddH 2O 60 ul total2. Gel purify digested plasmid using QIAquick Gel Extraction Kit and elute in EB. If BsmBI digested, a ~2kb filler piece should be present on the gel. Only gel purify the larger band . Leave the 2kb band.3. Phosphorylate and anneal each pair of oligos: 1 ul Oligo 1 (100 µM) 1 ul Oligo 2 (100 µM) 1 ul 10X T4 Ligation Buffer (NEB) 6.5 ul ddH 2O 0.5 ul T4 PNK (NEB M0201S) 10 ul total Please use the T4 Ligation Buffer since the buffer supplied with the T4 PNK enzyme does not include ATP (or supplement to 1mM ATP).Put the phosphorylation/annealing reaction in a thermocycler using the following parameters: 37o C 30 min 95o C 5 min and then ramp down to 25o C at 5o C/min4. Dilute annealed oligos from Step 3 at a 1:200 dilution into sterile water or EB.5. Set up ligation reaction and incubate at room temperature for 10 min:X ul BsmB I digested plasmidfrom Step 2 (50ng)1 ul diluted oligo duplex from Step 4 5 ul 2X Quick Ligase Buffer (NEB) X ul ddH 2O 10 ul subtotal1 ul Quick Ligase (NEB M2200S) 11 ul total Also perform a negative control ligation (vector-only with water in place of oligos) and transformation.6.Transformation into Stbl3 bacteria . Lentiviral transfer plasmids contain Long-Terminal Repeats (LTRs) and must be transformed into recombination-deficient bacteria. We use homemade Stbl3 (propagated from Invitrogen C7373-03) and get excellent plasmid yields. Although other RecA- strains may work, we have found the most consistent transformations and yields using Stbl3.。
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。
慢病毒生产及使用操作手册
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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@
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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
慢病毒使用操作指南一、慢病毒的储存与稀释：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）注：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。
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。
二、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. 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）以及在体内（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）2. 慢病毒感染目的细胞预实验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（HEK293T，Hela）细胞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。
如：病毒滴度为>1X108 TU/ml 即每毫升病毒液中至少含有1X108个具有生物活性的慢病毒颗粒。
②以24孔培养板为例，进行目的细胞和HEK293T 细胞的感染预实验实验前按照不同的MOI设置不同的感染孔，并根据MOI和细胞数量计算所需要的病毒量，如有必要可以使用PBS溶液或者无血清培养基稀释病毒原液第一天，准备细胞：在24孔培养板接种若干孔，每个孔内接种3~5X104个目的细胞，铺板时细胞的融合率为50%左右，每孔培养基体积为100 μl；进行病毒感染时细胞的融合度约为70%左右。
慢病毒载体包装构建过程
慢病毒载体包装构建过程原理：慢病毒载体可以将外源基因或外源的shRNA有效地整合到宿主染色体上，从而达到持久性表达目的序列的效果。
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、干细胞等多种类型的细胞，从而达到良好的的基因治疗效果。
对于一些较难转染的细胞, 如原代细胞、干细胞、不分化的细胞等，使用慢病毒载体,能大大提高目的基因或目的shRNA的转导效率，且目的基因或目的shRNA整合到宿主细胞基因组的几率大大增加，能够比较方便快捷地实现目的基因或目的shRNA的长期、稳定表达.概念：慢病毒载体是指以人类免疫缺陷病毒-1 （H IV-1) 来源的一种病毒载体，慢病毒载体包含了包装、转染、稳定整合所需要的遗传信息，是慢病毒载体系统的主要组成部分。
携带有外源基因的慢病毒载体在慢病毒包装质粒、细胞系的辅助下，经过病毒包装成为有感染力的病毒颗粒,通过感染细胞或活体组织，实现外源基因在细胞或活体组织中表达。
辅助成分：慢病毒载体辅助成分包括: 慢病毒包装质粒和可产生病毒颗粒的细胞系。
慢病毒载体包含了包装、转染、稳定整合所需要的遗传信息。
慢病毒包装质粒可提供所有的转录并包装RNA 到重组的假病毒载体所需要的所有辅助蛋白.为产生高滴度的病毒颗粒,需要利用表达载体和包装质粒同时共转染细胞,在细胞中进行病毒的包装，包装好的假病毒颗粒分泌到细胞外的培养基中，离心取得上清液后，可以直接用于宿主细胞的感染,目的基因进入到宿主细胞之后，经过反转录，整合到基因组,从而高水平的表达效应分子。
基本原理：慢病毒载体系统由两部分组成,即包装成分和载体成分.包装成分：由HIV—1基因组去除了包装、逆转录和整合所需的顺式作用序列而构建，能够反式提供产生病毒颗粒所必需的蛋白。
包装成分通常被分开构建到两个质粒上,一个质粒表达Gag和Pol蛋白,另一个质粒表达Env蛋白，其目的也是降低恢复成野生型病毒的可能.将包装成分与载体成分的3个质粒共转染细胞(如人肾293T细胞)，即可在细胞上清中收获只有一次性感染能力而无复制能力的、携带目的基因的HIV-1载体颗粒。
汉恒生物-慢病毒生产及使用操作手册第二版
2. 感染细胞最佳 MOI 的测定 MOI（Multiplicity of Infection，感染复数）是指每个细胞感染的病毒
数，通常 MOI 越高，病毒整合到染色体的数量以及目的蛋白的表达量越高。 对于分裂活跃的细胞，比如 Hela、293 细胞，MOI=1～3 时，80%以上的细胞均表 达目的基因。而对于非分裂细胞，比如原代细胞，感染效率较低。需要进行 MOI 梯度摸索实验，选择适合的 MOI 进行实验。
四、慢病毒包装和浓缩 （一）质粒扩增
构建好的慢病毒载体和辅助质粒需经过大量抽提，浓度大于 1ug/ul,A260/280 在 1.7-1.8 间方可用以包毒。推荐使用 Qiagen 大抽试剂盒进 行质粒的大量去内毒素抽提。 （二）传 293T 细胞
将培养 293T 细胞 T75 瓶中的培养基吸净，加入 2mL 4 度冰箱取出的 0.25% 胰酶，使其均匀覆盖瓶底，置于 37 度培养箱中 3-5min，取出，摇晃可发现细胞 于底部脱离，将其全部晃下，加入 3mL 37 度水浴中预热的 10％ DMEM，移液枪 用 10mL 移液管进行吹打，较大力吹打 6-8 次即可，不留死角，瓶口处较难吹打 可将移液管对准培口，小力将培养基打出即可覆盖到接近瓶口的细胞。之后，将 所有细胞吸出，置于 15mL 离心管中，取 50ul 混匀后的细胞于 1.5mL eppendorf
当细胞传代次数过多，细胞状态变差时，或者细胞出现污染事故时，需要丢 弃并对最初冻存的细胞进行复苏。
1、设置温度为 37~42℃的水浴。 2、查看细胞库记录，根据记录从液氮罐中取出冻存的细胞（需戴上棉手套， 防止被冻伤），迅速丢入水浴锅中并快速晃动，尽量在 1~2min 内使细胞溶液完全 溶解。 3、将细胞溶液转移到 15ml 离心管中，并在其中加上 1ml 新鲜的完全培养 基，混匀后离心，1000rpm，5min。 4、去掉上清，加入 5ml 新鲜的完全培养基，混匀沉淀后，转入 6cm 培养皿。 5、将培养皿平稳放入 37℃、5%CO2 和 95%相对湿度的培养箱中培养。 6、第二天观察细胞存活率。给细胞换一下培养基。以后每天观察细胞生长 情况。
pLVX-IRES-Neo慢病毒载体使用说明
pLVX-IRES-Neo pLVX-IRES-Neo载体基本信息：载体名称:pLVX-IRES-Neo, pLVX IRES Neo质粒类型: 慢病毒载体；哺乳动物细胞表达载体；双顺反子载体高拷贝/低拷贝: 高拷贝启动子: CMV克隆方法: 多克隆位点，限制性内切酶载体大小: 8269 bp5' 测序引物及序列: CMV-F: CGCAAATGGGCGGTAGGCGTG (Invitrogen)3' 测序引物及序列: IRES-R: CCTCACATTGCCAAAAGACG载体标签: 无标签载体抗性: 氨苄青霉素筛选标记: 新霉素Neomycin克隆菌株: Stbl3 E.coli备注: pLVX-IRES-Neo载体以双顺反子的形式同时表达Neo抗性基因和目的基因；CMV启动子驱动目的基因的过表达。
稳定性: 稳表达组成型: 组成型病毒/非病毒: 慢病毒pLVX-IRES-Neo载体质粒图谱和多克隆位点信息：pLVX-IRES-Neo载体序列：ORIGIN1 TGGAAGGGCT AATTCACTCC CAAAGAAGAC AAGATATCCT TGATCTGTGG ATCTACCACA61 CACAAGGCTA CTTCCCTGAT TAGCAGAACT ACACACCAGG GCCAGGGGTC AGATATCCAC121 TGACCTTTGG ATGGTGCTAC AAGCTAGTAC CAGTTGAGCC AGATAAGGTA GAAGAGGCCA 181 ATAAAGGAGA GAACACCAGC TTGTTACACC CTGTGAGCCT GCATGGGATG GATGACCCGG 241 AGAGAGAAGT GTTAGAGTGG AGGTTTGACA GCCGCCTAGC ATTTCATCAC GTGGCCCGAG 301 AGCTGCATCC GGAGTACTTC AAGAACTGCT GATATCGAGC TTGCTACAAG GGACTTTCCG 361 CTGGGGACTT TCCAGGGAGG CGTGGCCTGG GCGGGACTGG GGAGTGGCGA GCCCTCAGAT 421 CCTGCATATA AGCAGCTGCT TTTTGCCTGT ACTGGGTCTC TCTGGTTAGA CCAGATCTGA481 GCCTGGGAGC TCTCTGGCTA ACTAGGGAAC CCACTGCTTA AGCCTCAATA AAGCTTGCCT 541 TGAGTGCTTC AAGTAGTGTG TGCCCGTCTG TTGTGTGACT CTGGTAACTA GAGATCCCTC601 AGACCCTTTT AGTCAGTGTG GAAAATCTCT AGCAGTGGCG CCCGAACAGG GACTTGAAAG 661 CGAAAGGGAA ACCAGAGGAG CTCTCTCGAC GCAGGACTCG GCTTGCTGAA GCGCGCACGG 721 CAAGAGGCGA GGGGCGGCGA CTGGTGAGTA CGCCAAAAAT TTTGACTAGC GGAGGCTAGA 781 AGGAGAGAGA TGGGTGCGAG AGCGTCAGTA TTAAGCGGGG GAGAATTAGA TCGCGATGGG 841 AAAAAATTCG GTTAAGGCCA GGGGGAAAGA AAAAATATAA ATTAAAACAT ATAGTATGGG 901 CAAGCAGGGA GCTAGAACGA TTCGCAGTTA ATCCTGGCCT GTTAGAAACA TCAGAAGGCT 961 GTAGACAAAT ACTGGGACAG CTACAACCAT CCCTTCAGAC AGGATCAGAA GAACTTAGAT 1021 CATTATATAA TACAGTAGCA ACCCTCTATT GTGTGCATCA AAGGATAGAG ATAAAAGACA 1081 CCAAGGAAGC TTTAGACAAG ATAGAGGAAG AGCAAAACAA AAGTAAGACC ACCGCACAGC 1141 AAGCGGCCGG CCGCTGATCT TCAGACCTGG AGGAGGAGAT ATGAGGGACA ATTGGAGAAG 1201 TGAATTATAT AAATATAAAG TAGTAAAAAT TGAACCATTA GGAGTAGCAC CCACCAAGGC 1261 AAAGAGAAGA GTGGTGCAGA GAGAAAAAAG AGCAGTGGGA ATAGGAGCTT TGTTCCTTGG 1321 GTTCTTGGGA GCAGCAGGAA GCACTATGGG CGCAGCGTCA ATGACGCTGA CGGTACAGGC 1381 CAGACAATTA TTGTCTGGTA TAGTGCAGCA GCAGAACAAT TTGCTGAGGG CTATTGAGGC 1441 GCAACAGCAT CTGTTGCAAC TCACAGTCTG GGGCATCAAG CAGCTCCAGG CAAGAATCCT1501 GGCTGTGGAA AGATACCTAA AGGATCAACA GCTCCTGGGG ATTTGGGGTT GCTCTGGAAA 1561 ACTCATTTGC ACCACTGCTG TGCCTTGGAA TGCTAGTTGG AGTAATAAAT CTCTGGAACA 1621 GATTTGGAAT CACACGACCT GGATGGAGTG GGACAGAGAA ATTAACAATT ACACAAGCTT 1681 AATACACTCC TTAATTGAAG AATCGCAAAA CCAGCAAGAA AAGAATGAAC AAGAATTATT 1741 GGAATTAGAT AAATGGGCAA GTTTGTGGAA TTGGTTTAAC ATAACAAATT GGCTGTGGTA 1801 TATAAAATTA TTCATAATGA TAGTAGGAGG CTTGGTAGGT TTAAGAATAG TTTTTGCTGT 1861 ACTTTCTATA GTGAATAGAG TTAGGCAGGG ATATTCACCA TTATCGTTTC AGACCCACCT 1921 CCCAACCCCG AGGGGACCCG ACAGGCCCGA AGGAATAGAA GAAGAAGGTG GAGAGAGAGA 1981 CAGAGACAGA TCCATTCGAT TAGTGAACGG ATCTCGACGG TATCGCCTTT AAAAGAAAAG 2041 GGGGGATTGG GGGGTACAGT GCAGGGGAAA GAATAGTAGA CATAATAGCA ACAGACATAC 2101 AAACTAAAGA ATTACAAAAA CAAATTACAA AAATTCAAAA TTTTCGGGTT TATTACAGGG 2161 ACAGCAGAGA TCCAGTTTAT CGATAAGCTT GGGAGTTCCG CGTTACATAA CTTACGGTAA 2221 ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG 2281 TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT 2341 AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC AAGTACGCCC CCTATTGACG 2401 TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA CATGACCTTA TGGGACTTTC 2461 CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC 2521 AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA 2581 TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG GGACTTTCCA AAATGTCGTA 2641 ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT ACGGTGGGAG GTCTATATAA 2701 GCAGAGCTCG TTTAGTGAAC CGTCAGATCG CCTGGAGACG CCATCCACGC TGTTTTGACC 2761 TCCATAGAAG ACACCGACTC TACTAGAGGA TCTATTTCCG GTGAATTCCT CGAGACTAGT 2821 TCTAGAGCGG CCGCGGATCC CGCCCCTCTC CCTCCCCCCC CCCTAACGTT ACTGGCCGAA 2881 GCCGCTTGGA ATAAGGCCGG TGTGCGTTTG TCTATATGTT ATTTTCCACC ATATTGCCGT 2941 CTTTTGGCAA TGTGAGGGCC CGGAAACCTG GCCCTGTCTT CTTGACGAGC ATTCCTAGGG 3001 GTCTTTCCCC TCTCGCCAAA GGAATGCAAG GTCTGTTGAA TGTCGTGAAG GAAGCAGTTC 3061 CTCTGGAAGC TTCTTGAAGA CAAACAACGT CTGTAGCGAC CCTTTGCAGG CAGCGGAACC 3121 CCCCACCTGG CGACAGGTGC CTCTGCGGCC AAAAGCCACG TGTATAAGAT ACACCTGCAA 3181 AGGCGGCACA ACCCCAGTGC CACGTTGTGA GTTGGATAGT TGTGGAAAGA GTCAAATGGC 3241 TCTCCTCAAG CGTATTCAAC AAGGGGCTGA AGGATGCCCA GAAGGTACCC CATTGTATGG 3301 GATCTGATCT GGGGCCTCGG TGCACATGCT TTACATGTGT TTAGTCGAGG TTAAAAAAAC 3361 GTCTAGGCCC CCCGAACCAC GGGGACGTGG TTTTCCTTTG AAAAACACGA TGATAAGCTT 3421 GCCACAACCA TGGCTGAACA AGATGGATTG CACGCAGGTT CTCCGGCCGC TTGGGTGGAG 3481 AGGCTATTCG GCTATGACTG GGCACAACAG ACAATCGGCT GCTCTGATGC CGCCGTGTTC 3541 CGGCTGTCAG CGCAGGGGCG CCCGGTTCTT TTTGTCAAGA CCGACCTGTC CGGTGCCCTG 3601 AATGAACTGC AGGACGAGGC AGCGCGGCTA TCGTGGCTGG CCACGACGGG CGTTCCTTGC 3661 GCAGCTGTGC TCGACGTTGT CACTGAAGCG GGAAGGGACT GGCTGCTATT GGGCGAAGTG 3721 CCGGGGCAGG ATCTCCTGTC ATCTCACCTT GCTCCTGCCG AGAAAGTATC CATCATGGCT 3781 GATGCAATGC GGCGGCTGCA TACGCTTGAT CCGGCTACCT GCCCATTCGA CCACCAAGCG 3841 AAACATCGCA TCGAGCGAGC ACGTACTCGG ATGGAAGCCG GTCTTGTCGA TCAGGATGAT 3901 CTGGACGAAG AGCATCAGGG GCTCGCGCCA GCCGAACTGT TCGCCAGGCT CAAGGCGCGC 3961 ATGCCCGACG GCGAGGATCT CGTCGTGACC CATGGCGATG CCTGCTTGCC GAATATCATG 4021 GTGGAAAATG GCCGCTTTTC TGGATTCATC GACTGTGGCC GGCTGGGTGT GGCGGACCGC 4081 TATCAGGACA TAGCGTTGGC TACCCGTGAT ATTGCTGAAG AGCTTGGCGG CGAATGGGCT4141 GACCGCTTCC TCGTGCTTTA CGGTATCGCC GCTCCCGATT CGCAGCGCAT CGCCTTCTAT 4201 CGCCTTCTTG ACGAGTTCTT CTGAACGCGT CTGGAACAAT CAACCTCTGG ATTACAAAAT 4261 TTGTGAAAGA TTGACTGGTA TTCTTAACTA TGTTGCTCCT TTTACGCTAT GTGGATACGC 4321 TGCTTTAATG CCTTTGTATC ATGCTATTGC TTCCCGTATG GCTTTCATTT TCTCCTCCTT4381 GTATAAATCC TGGTTGCTGT CTCTTTATGA GGAGTTGTGG CCCGTTGTCA GGCAACGTGG 4441 CGTGGTGTGC ACTGTGTTTG CTGACGCAAC CCCCACTGGT TGGGGCATTG CCACCACCTG 4501 TCAGCTCCTT TCCGGGACTT TCGCTTTCCC CCTCCCTATT GCCACGGCGG AACTCATCGC 4561 CGCCTGCCTT GCCCGCTGCT GGACAGGGGC TCGGCTGTTG GGCACTGACA ATTCCGTGGT 4621 GTTGTCGGGG AAGCTGACGT CCTTTCCATG GCTGCTCGCC TGTGTTGCCA CCTGGATTCT 4681 GCGCGGGACG TCCTTCTGCT ACGTCCCTTC GGCCCTCAAT CCAGCGGACC TTCCTTCCCG 4741 CGGCCTGCTG CCGGCTCTGC GGCCTCTTCC GCGTCTTCGC CTTCGCCCTC AGACGAGTCG 4801 GATCTCCCTT TGGGCCGCCT CCCCGCCTGG AATTAATTCT GCAGTCGAGA CCTAGAAAAA 4861 CATGGAGCAA TCACAAGTAG CAATACAGCA GCTACCAATG CTGATTGTGC CTGGCTAGAA 4921 GCACAAGAGG AGGAGGAGGT GGGTTTTCCA GTCACACCTC AGGTACCTTT AAGACCAATG 4981 ACTTACAAGG CAGCTGTAGA TCTTAGCCAC TTTTTAAAAG AAAAGAGGGG ACTGGAAGGG 5041 CTAATTCACT CCCAACGAAG ACAAGATATC CTTGATCTGT GGATCTACCA CACACAAGGC 5101 TACTTCCCTG ATTAGCAGAA CTACACACCA GGGCCAGGGG TCAGATATCC ACTGACCTTT 5161 GGATGGTGCT ACAAGCTAGT ACCAGTTGAG CCAGATAAGG TAGAAGAGGC CAATAAAGGA 5221 GAGAACACCA GCTTGTTACA CCCTGTGAGC CTGCATGGGA TGGATGACCC GGAGAGAGAA 5281 GTGTTAGAGT GGAGGTTTGA CAGCCGCCTA GCATTTCATC ACGTGGCCCG AGAGCTGCAT 5341 CCGGAGTACT TCAAGAACTG CTGATATCGA GCTTGCTACA AGGGACTTTC CGCTGGGGAC 5401 TTTCCAGGGA GGCGTGGCCT GGGCGGGACT GGGGAGTGGC GAGCCCTCAG ATCCTGCATA 5461 TAAGCAGCTG CTTTTTGCCT GTACTGGGTC TCTCTGGTTA GACCAGATCT GAGCCTGGGA 5521 GCTCTCTGGC TAACTAGGGA ACCCACTGCT TAAGCCTCAA TAAAGCTTGC CTTGAGTGCT 5581 TCAAGTAGTG TGTGCCCGTC TGTTGTGTGA CTCTGGTAAC TAGAGATCCC TCAGACCCTT 5641 TTAGTCAGTG TGGAAAATCT CTAGCAGTAG TAGTTCATGT CATCTTATTA TTCAGTATTT 5701 ATAACTTGCA AAGAAATGAA TATCAGAGAG TGAGAGGCCT TGACATTGCT AGCGTTTACC 5761 GTCGACCTCT AGCTAGAGCT TGGCGTAATC ATGGTCATAG CTGTTTCCTG TGTGAAATTG 5821 TTATCCGCTC ACAATTCCAC ACAACATACG AGCCGGAAGC ATAAAGTGTA AAGCCTGGGG 5881 TGCCTAATGA GTGAGCTAAC TCACATTAAT TGCGTTGCGC TCACTGCCCG CTTTCCAGTC 5941 GGGAAACCTG TCGTGCCAGC TGCATTAATG AATCGGCCAA CGCGCGGGGA GAGGCGGTTT 6001 GCGTATTGGG CGCTCTTCCG CTTCCTCGCT CACTGACTCG CTGCGCTCGG TCGTTCGGCT 6061 GCGGCGAGCG GTATCAGCTC ACTCAAAGGC GGTAATACGG TTATCCACAG AATCAGGGGA 6121 TAACGCAGGA AAGAACATGT GAGCAAAAGG CCAGCAAAAG GCCAGGAACC GTAAAAAGGC 6181 CGCGTTGCTG GCGTTTTTCC ATAGGCTCCG CCCCCCTGAC GAGCATCACA AAAATCGACG 6241 CTCAAGTCAG AGGTGGCGAA ACCCGACAGG ACTATAAAGA TACCAGGCGT TTCCCCCTGG 6301 AAGCTCCCTC GTGCGCTCTC CTGTTCCGAC CCTGCCGCTT ACCGGATACC TGTCCGCCTT 6361 TCTCCCTTCG GGAAGCGTGG CGCTTTCTCA TAGCTCACGC TGTAGGTATC TCAGTTCGGT 6421 GTAGGTCGTT CGCTCCAAGC TGGGCTGTGT GCACGAACCC CCCGTTCAGC CCGACCGCTG 6481 CGCCTTATCC GGTAACTATC GTCTTGAGTC CAACCCGGTA AGACACGACT TATCGCCACT 6541 GGCAGCAGCC ACTGGTAACA GGATTAGCAG AGCGAGGTAT GTAGGCGGTG CTACAGAGTT 6601 CTTGAAGTGG TGGCCTAACT ACGGCTACAC TAGAAGAACA GTATTTGGTA TCTGCGCTCT 6661 GCTGAAGCCA GTTACCTTCG GAAAAAGAGT TGGTAGCTCT TGATCCGGCA AACAAACCAC 6721 CGCTGGTAGC GGTGGTTTTT TTGTTTGCAA GCAGCAGATT ACGCGCAGAA AAAAAGGATC6781 TCAAGAAGAT CCTTTGATCT TTTCTACGGG GTCTGACGCT CAGTGGAACG AAAACTCACG6841 TTAAGGGATT TTGGTCATGA GATTATCAAA AAGGATCTTC ACCTAGATCC TTTTAAATTA6901 AAAATGAAGT TTTAAATCAA TCTAAAGTAT ATATGAGTAA ACTTGGTCTG ACAGTTACCA6961 ATGCTTAATC AGTGAGGCAC CTATCTCAGC GATCTGTCTA TTTCGTTCAT CCATAGTTGC7021 CTGACTCCCC GTCGTGTAGA TAACTACGAT ACGGGAGGGC TTACCATCTG GCCCCAGTGC7081 TGCAATGATA CCGCGAGACC CACGCTCACC GGCTCCAGAT TTATCAGCAA TAAACCAGCC7141 AGCCGGAAGG GCCGAGCGCA GAAGTGGTCC TGCAACTTTA TCCGCCTCCA TCCAGTCTAT7201 TAATTGTTGC CGGGAAGCTA GAGTAAGTAG TTCGCCAGTT AATAGTTTGC GCAACGTTGT7261 TGCCATTGCT ACAGGCATCG TGGTGTCACG CTCGTCGTTT GGTATGGCTT CATTCAGCTC7321 CGGTTCCCAA CGATCAAGGC GAGTTACATG ATCCCCCATG TTGTGCAAAA AAGCGGTTAG7381 CTCCTTCGGT CCTCCGATCG TTGTCAGAAG TAAGTTGGCC GCAGTGTTAT CACTCATGGT7441 TATGGCAGCA CTGCATAATT CTCTTACTGT CATGCCATCC GTAAGATGCT TTTCTGTGAC7501 TGGTGAGTAC TCAACCAAGT CATTCTGAGA ATAGTGTATG CGGCGACCGA GTTGCTCTTG7561 CCCGGCGTCA ATACGGGATA ATACCGCGCC ACATAGCAGA ACTTTAAAAG TGCTCATCAT7621 TGGAAAACGT TCTTCGGGGC GAAAACTCTC AAGGATCTTA CCGCTGTTGA GATCCAGTTC7681 GATGTAACCC ACTCGTGCAC CCAACTGATC TTCAGCATCT TTTACTTTCA CCAGCGTTTC7741 TGGGTGAGCA AAAACAGGAA GGCAAAATGC CGCAAAAAAG GGAATAAGGG CGACACGGAA7801 ATGTTGAATA CTCATACTCT TCCTTTTTCA ATATTATTGA AGCATTTATC AGGGTTATTG7861 TCTCATGAGC GGATACATAT TTGAATGTAT TTAGAAAAAT AAACAAATAG GGGTTCCGCG7921 CACATTTCCC CGAAAAGTGC CACCTGACGT CGACGGATCG GGAGATCAAC TTGTTTATTG7981 CAGCTTATAA TGGTTACAAA TAAAGCAATA GCATCACAAA TTTCACAAAT AAAGCATTTT8041 TTTCACTGCA TTCTAGTTGT GGTTTGTCCA AACTCATCAA TGTATCTTAT CATGTCTGGA8101 TCAACTGGAT AACTCAAGCT AACCAAAATC ATCCCAAACT TCCCACCCCA TACCCTATTA8161 CCACTGCCAA TTACCTGTGG TTTCATTTAC TCTAAACCTG TGATTCCTCT GAATTATTTT8221 CATTTTAAAG AAATTGTATT TGTTAAATAT GTACTACAAA CTTAGTAGT//pLVX-IRES-Neo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。
慢病毒包装操作方案
慢病毒包装操作流程一、材料1 细胞培养试剂试剂名称终浓度试剂品牌DMEM/High glucose基础培养基90%Invitrogen胎牛血清10%Invitrogen/Gibco丙酮酸钠1mM Invitrogen2 慢病毒包装试剂试剂名称浓度试剂品牌Lipofectamine 2000InvitrogenPEG6000溶液50%WakoHBSS溶液Invitrogen3 耗材50 mL离心管15 mL离心管10 cm细胞培养皿μm过滤器2 mL EP管二、操作流程1细胞培养1.1293T/293FT细胞的复苏1)将完全培养液从4°C中取出放置到室温预热30 min左右。
在超净台内，用吸管吸取6~7mL完全培养液至15 mL离心管中；2)快速将冻存的细胞从液氮中取出，并迅速用镊子夹住盖子放入37°C 水浴中快速晃动（水不要没到盖子），使其在1~2分钟内完全融化；3)在超净台内，用酒精棉球擦拭冻存管外壁消毒，用吸管吸取所有融化的细胞悬液至装准备好的完全培养液中，轻轻吹打混匀，使冻存液分散开（目的是让DMSO分散，降低恢复室温的DMSO对细胞造成的毒性作用）。
4)在室温条件下，250 g离心4分钟。
5)离心后，在超净台内小心倒去上清，用吸管吸取2 mL新鲜完全培养液重悬细胞至单细胞悬液，再转移已经加好培养基的培养瓶/培养皿中，写上细胞名称、日期，放置37°C、5% CO2饱和湿度培养箱内培养。
（首次复苏细胞时，离心重悬后需取样计数，根据细胞数选择面积合适的培养容器。
）6)复苏翌日，给复苏的293T细胞更换新鲜的完全培养基。
1.2293T/293FT细胞传代1)待细胞长至60%-70%融合度即可传代。
将培养瓶里的所有培养液全部移去，用1×PBS洗涤细胞两次（洗涤速度要快，避免细胞干涸时间过长），以去除残余的培养液和血清（血清含有胰酶的抑制因子）；2)加入适当的胰酶溶液，能使其完全浸过细胞即可，室温孵育1-2分钟。
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pLOX-Ttag-iresTK pLOX-Ttag-iresTK载体基本信息：载体名称:pLOX-Ttag-iresTK质粒类型: 慢病毒系统载体高拷贝/低拷贝: 高拷贝启动子: --克隆方法: 多克隆位点，限制性内切酶载体大小: 12292bp5' 测序引物及序列: CMV Fwd3' 测序引物及序列: SP6 rev载体标签: --载体抗性: 氨苄青霉素（Ampicillian）筛选标记: --备注: 克隆菌株 Stbl3 或HB101，以减少基因重组现象的发生。
稳定性: --组成型: --病毒/非病毒: 慢病毒pLOX-Ttag-iresTK载体质粒图谱和多克隆位点信息：pLOX-Ttag-iresTK载体序列ORIGIN1 AGTTGGAAGG GCTAATTCAC TCCCAAAGAA GACAAGATAT CCTTGATCTG TGGATCTACC61 ACACACAAGG CTACTTCCCT GATTAGCAGA ACTACACACC AGGGCCAGGG GTCAGATATC 121 CACTGACCTT TGGATGGTGC TACAAGCTAG TACCAGTTGA GCCAGATAAG GTAGAAGAGG 181 CCAATAAAGG AGAGAACACC AGCTTGTTAC ACCCTGTGAG CCTGCATGGG ATGGATGACC 241 CGGAGAGAGA AGTGTTAGAG TGGAGGTTTG ACAGCCGCCT AGCATTTCAT CACGTGGCCC 301 GAGAGCTGCA TCCGGAGTAC TTCAAGAACT GCTGATATCG AGCTTGCTAC AAGGGACTTT 361 CCGCTGGGGA CTTTCCAGGG AGGCGTGGCC TGGGCGGGAC TGGGGAGTGG CGAGCCCTCA 421 GATCCTGCAT ATAAGCAGCT GCTTTTTGCC TGTACTGGGT CTCTCTGGTT AGACCAGATC 481 TGAGCCTGGG AGCTCTCTGG CTAACTAGGG AACCCACTGC TTAAGCCTCA ATAAAGCTTG 541 CCTTGAGTGC TTCAAGTAGT GTGTGCCCGT CTGTTGTGTG ACTCTGGTAA CTAGAGATCC 601 CTCAGACCCT TTTAGTCAGT GTGGAAAATC TCTAGCAGTG GCGCCCGAAC AGGGACTTGA 661 AAGCGAAAGG GAAACCAGAG GAGCTCTCTC GACGCAGGAC TCGGCTTGCT GAAGCGCGCA 721 CGGCAAGAGG CGAGGGGCGG CGACTGGTGA GTACGCCAAA AATTTTGACT AGCGGAGGCT 781 AGAAGGAGAG AGATGGGTGC GAGAGCGTCA GTATTAAGCG GGGGAGAATT AGATCGCGAT 841 GGGAAAAAAT TCGGTTAAGG CCAGGGGGAA AGAAAAAATA TAAATTAAAA CATATAGTAT 901 GGGCAAGCAG GGAGCTAGAA CGATTCGCAG TTAATCCTGG CCTGTTAGAA ACATCAGAAG 961 GCTGTAGACA AATACTGGGA CAGCTACAAC CATCCCTTCA GACAGGATCA GAAGAACTTA 1021 GATCATTATA TAATACAGTA GCAACCCTCT ATTGTGTGCA TCAAAGGATA GAGATAAAAG 1081 ACACCAAGGA AGCTTTAGAC AAGATAGAGG AAGAGCAAAA CAAAAGTAAG ACCACCGCAC 1141 AGCAAGCGGC CGCTGATCTT CAGACCTGGA GGAGGAGATA TGAGGGACAA TTGGAGAAGT 1201 GAATTATATA AATATAAAGT AGTAAAAATT GAACCATTAG GAGTAGCACC CACCAAGGCA 1261 AAGAGAAGAG TGGTGCAGAG AGAAAAAAGA GCAGTGGGAA TAGGAGCTTT GTTCCTTGGG 1321 TTCTTGGGAG CAGCAGGAAG CACTATGGGC GCAGCGTCAA TGACGCTGAC GGTACAGGCC 1381 AGACAATTAT TGTCTGGTAT AGTGCAGCAG CAGAACAATT TGCTGAGGGC TATTGAGGCG 1441 CAACAGCATC TGTTGCAACT CACAGTCTGG GGCATCAAGC AGCTCCAGGC AAGAATCCTG 1501 GCTGTGGAAA GATACCTAAA GGATCAACAG CTCCTGGGGA TTTGGGGTTG CTCTGGAAAA 1561 CTCATTTGCA CCACTGCTGT GCCTTGGAAT GCTAGTTGGA GTAATAAATC TCTGGAACAG 1621 ATTTGGAATC ACACGACCTG GATGGAGTGG GACAGAGAAA TTAACAATTA CACAAGCTTA 1681 ATACACTCCT TAATTGAAGA ATCGCAAAAC CAGCAAGAAA AGAATGAACA AGAATTATTG 1741 GAATTAGATA AATGGGCAAG TTTGTGGAAT TGGTTTAACA TAACAAATTG GCTGTGGTAT 1801 ATAAAATTAT TCATAATGAT AGTAGGAGGC TTGGTAGGTT TAAGAATAGT TTTTGCTGTA 1861 CTTTCTATAG TGAATAGAGT TAGGCAGGGA TATTCACCAT TATCGTTTCA GACCCACCTC 1921 CCAACCCCGA GGGGACCCGA CAGGCCCGAA GGAATAGAAG AAGAAGGTGG AGAGAGAGAC 1981 AGAGACAGAT CCATTCGATT AGTGAACGGA TCTCGACGGT ATCGATAAGC TTGGGAGTTC 2041 CGCGTTACAT AACTTACGGT AAATGGCCCG CCTGGCTGAC CGCCCAACGA CCCCCGCCCA 2101 TTGACGTCAA TAATGACGTA TGTTCCCATA GTAACGCCAA TAGGGACTTT CCATTGACGT 2161 CAATGGGTGG AGTATTTACG GTAAACTGCC CACTTGGCAG TACATCAAGT GTATCATATG 2221 CCAAGTACGC CCCCTATTGA CGTCAATGAC GGTAAATGGC CCGCCTGGCA TTATGCCCAG 2281 TACATGACCT TACGGGACTT TCCTACTTGG CAGTACATCT ACGTATTAGT CATCGCTATT 2341 ACCATGGTGA TGCGGTTTTG GCAGTACACC AATGGGCGTG GATAGCGGTT TGACTCACGG 2401 GGATTTCCAA GTCTCCACCC CATTGACGTC AATGGGAGTT TGTTTTGGCA CCAAAATCAA 2461 CGGGACTTTC CAAAATGTCG TAACAACTCC GCCCCATTGA CGCAAATGGG CGGTAGGCGT2521 GTACGGTGGG AGGTCTATAT AAGCAGAGCT CGTTTAGTGA ACCGTCAGAT CGCCTGGAGA 2581 CGCCATCCAC GCTGTTTTGA CCTCCATAGA AGACACCGAC TCTAGCTAGA GGATCCCCCG 2641 GGCTGCAGGA ATTCCCGGCC TCTGAGCTAT TCCAGAAGTA GTGAAGAGGC TTTTTTGGAG 2701 GCCTAGGCTT TTGCAAAAAG CTTTGCAAAG ATGGATAAAG TTTTAAACAG AGAGGAATCT 2761 TTGCAGCTAA TGGACCTTCT AGGTCTTGAA AGGAGTGCCT GGGGGAATAT TCCTCTGATG 2821 AGAAAGGCAT ATTTAAAAAA ATGCAAGGAG TTTCATCCTG ATAAAGGAGG AGATGAAGAA 2881 AAAATGAAGA AAATGAATAC TCTGTACAAG AAAATGGAAG ATGGAGTAAA ATATGCTCAT 2941 CAACCTGACT TTGGAGGCTT CTGGGATGCA ACTGAGGTAT TTGCTTCTTC CTTAAATCCT 3001 GGTGTTGATG CAATGTACTG CAAACAATGG CCTGAGTGTG CAAAGAAAAT GTCTGCTAAC 3061 TGCATATGCT TGCTGTGCTT ACTGAGGATG AAGCATGAAA ATAGAAAATT ATACAGGAAA 3121 GATCCACTTG TGTGGGTTGA TTGCTACTGC TTCGATTGCT TTAGAATGTG GTTTGGACTT 3181 GATCTTTGTG AAGGAACCTT ACTTCTGTGG TGTGACATAA TTGGACAAAC TACCTACAGA 3241 GATTTAAAGC TCTAAGGTAA ATATAAAATT TTTAAGTGTA TAATGTGTTA AACTACTGAT 3301 TCTAATTGTT TGTGTATTTT AGATTCCAAC CTATGGAACT GATGAATGGG AGCAGTGGTG 3361 GAATGCCTTT AATGAGGAAA ACCTGTTTTG CTCAGAAGAA ATGCCATCTA GTGATGATGA 3421 GGCTACTGCT GACTCTCAAC ATTCTACTCC TCCAAAAAAG AAGAGAAAGG TAGAAGACCC 3481 CAAGGACTTT CCTTCAGAAT TGCTAAGTTT TTTGAGTCAT GCTGTGTTTA GTAATAGAAC 3541 TCTTGCTTGC TTTGCTATTT ACACCACAAA GGAAAAAGCT GCACTGCTAT ACAAGAAAAT 3601 TATGGAAAAA TATTCTGTAA CCTTTATAAG TAGGCATAAC AGTTATAATC ATAACATACT 3661 GTTTTTTCTT ACTCCACACA GGCATAGAGT GTCTGCTATT AATAACTATG CTCAAAAATT 3721 GTGTACCTTT AGCTTTTTAA TTTGTAAAGG GGTTAATAAG GAATATTTGA TGTATAGTGC 3781 CTTGACTAGA GATCCATTTT CTGTTATTGA GGAAAGTTTG CCAGGTGGGT TAAAGGAGCA 3841 TGATTTTAAT CCAGAAGAAG CAGAGGAAAC TAAACAAGTG TCCTGGAAGC TTGTAACAGA 3901 GTATGCAATG GAAACAAAAT GTGATGATGT GTTGTTATTG CTTGGGATGT ACTTGGAATT 3961 TCAGTACAGT TTTGAAATGT GTTTAAAATG TATTAAAAAA GAACAGCCCA GCCACTATAA 4021 GTACCATGAA AAGCATTATG CAAATGCTGC TATATTTGCT GACAGCAAAA ACCAAAAAAC 4081 CATATGCCAA CAGGCTGTTG ATACTGTTTT AGCTAAAAAG CGGGTTGATA GCCTACAATT 4141 AACTAGAGAA CAAATGTTAA CAAACAGATT TAATGATCTT TTGGATAGGA TGGATATAAT 4201 GTTTGGTTCT ACAGGCTCTG CTGACATAGA AGAATGGATG GCTGGAGTTG CTTGGCTACA 4261 CTGTTTGTTG CCCAAAATGG ATTCAGTGGT GTATGACTTT TTAAAATGCA TGGTGTACAA 4321 CATTCCTAAA AAAAGATACT GGCTGTTTAA AGGACCAATT GATAGTGGTA AAACTACATT 4381 AGCAGCTGCT TTGCTTGAAT TATGTGGGGG GAAAGCTTTA AATGTTAATT TGCCCTTGGA 4441 CAGGCTGAAC TTTGAGCTAG GAGTAGCTAT TGACCAGTTT TTAGTAGTTT TTGAGGATGT 4501 AAAGGGCACT GGAGGGGAGT CCAGAGATTT GCCTTCAGGT CAGGGAATTA ATAACCTGGA 4561 CAATTTAAGG GATTATTTGG ATGGCAGTGT TAAGGTAAAC TTAGAAAAGA AACACCTAAA 4621 TAAAAGAACT CAAATATTTC CCCCTGGAAT AGTCACCATG AATGAGTACA GTGTGCCTAA 4681 AACACTGCAG GCCAGATTTG TAAAACAAAT AGATTTTAGG CCCAAAGATT ATTTAAAGCA 4741 TTGCCTGGAA CGCAGTGAGT TTTTGTTAGA AAAGAGAATA ATTCAAAGTG GCATTGCTTT 4801 GCTTCTTATG TTAATTTGGT ACAGACCTGT GGCTGAGTTT GCTCAAAGTA TTCAGAGCAG 4861 AATTGTGGAG TGGAAAGAGA GATTGGACAA AGAGTTTAGT TTGTCAGTGT ATCAAAAAAT 4921 GAAGTTTAAT GTGGCTATGG GAATTGGAGT TTTAGATTGG CTAAGAAACA GTGATGATGA 4981 TGATGAAGAC AGCCAGGAAA ATGCTGATAA AAATGAAGAT GGTGGGGAGA AGAACATGGA 5041 AGACTCAGGG CATGAAACAG GCATTGATTC ACAGTCCCAA GGCTCATTTC AGGCCCCTCA 5101 GTCCTCACAG TCTGTTCATG ATCATAATCA GCCATACCAC ATTTGTAGAG GTTTTACTTG5161 CTTTAAAAAA CCTCCCACAC CTCCCCCTGA ACCTGAAACA TAAAATGAAT GCAATTGTTG 5221 TTGTTAGCGG CCCCGGAATT CGATATCAAG CTTATCGATA CCGTCGACCT CGACATTAAT 5281 TCCGGTTATT TTCCACCATA TTGCCGTCTT TTGGCAATGT GAGGGCCCGG AAACCTGGCC 5341 CTGTCTTCTT GACGAGCATT CCTAGGGGTC TTTCCCCTCT CGCCAAAGGA ATGCAAGGTC 5401 TGTTGAATGT CGTGAAGGAA GCAGTTCCTC TGGAAGCTTC TTGAAGACAA ACAACGTCTG 5461 TAGCGACCCT TTGCAGGCAG CGGAACCCCC CACCTGGCGA CAGGTGCCTC TGCGGCCAAA 5521 AGCCACGTGT ATAAGATACA CCTGCAAAGG CGGCACAACC CCAGTGCCAC GTTGTGAGTT 5581 GGATAGTTGT GGAAAGAGTC AAATGGCTCT CCTCAAGCGT ATTCAACAAG GGGCTGAAGG 5641 ATGCCCAGAA GGTACCCCAT TGTATGGGAT CTGATCTGGG GCCTCGGTGC ACATGCTTTA 5701 CATGTGTTTA GTCGAGGTTA AAAAACGTCT AGGCCCCCCG AACCACGGGG ACGTGGTTTT 5761 CCTTTGAAAA ACACGATGAT AATACCATGG CTTCGTACCC CTGCCATCAA CACGCGTCTG 5821 CGTTCGACCA GGCTGCGCGT TCTCGCGGCC ATAACAACCG ACGTACGGCG TTGCGCCCTC 5881 GCCGGCAACA AAAAGCCACG GAAGTCCGCC TGGAGCAGAA AATGCCCACG CTACTGCGGG 5941 TTTATATAGA CGGTCCCCAC GGGATGGGGA AAACCACCAC CACGCAACTG CTGGTGGCCC 6001 TGGGTTCGCG CGACGATATC GTCTACGTAC CCGAGCCGAT GACTTACTGG CGGGTGTTGG 6061 GGGCTTCCGA GACAATCGCG AACATCTACA CCACACAACA CCGCCTCGAC CAGGGTGAGA 6121 TATCGGCCGG GGACGCGGCG GTGGTAATGA CAAGCGCCCA GATAACAATG GGCATGCCTT 6181 ATGCCGTGAC CGACGCCGTT CTGGCTCCTC ATATCGGGGG GGAGGCTGGG AGCTCACATG 6241 CCCCGCCCCC GGCCCTCACC CTCATCTTCG ACCGCCATCC CATCGCCGCC CTCCTGTGCT 6301 ACCCGGCCGC GCGATACCTT ATGGGCAGCA TGACCCCCCA GGCCGTGCTG GCGTTCGTGG 6361 CCCTCATCCC GCCGACCTTG CCCGGCACAA ACATCGTGTT GGGGGCCCTT CCGGAGGACA 6421 GACACATCGA CCGCCTGGCC AAACGCCAGC GCCCCGGCGA GCGGCTTGAC CTGGCTATGC 6481 TGGCCGCGTC GCCGCGTTTA TGGGCTGCTT GCCAATACGG TGCGGTATCT GCAGGGCGGC 6541 GGGTCGTGGC GGGAGGATTG GGGACAGCTT TCGGGGGCGG CCGTGCCGCC CCAGGGTGCC 6601 GAGCCCCAGA GCAACGCGGG CCCACGACCC CATATCGGGG ACACGTTATT TACCCTGTTT 6661 CGGGCCCCCG AGTTGCTGGC CCCCAACGGC GACCTGTATA ACGTGTTTGC CTGGGCTTTG 6721 GCTCGACGGT ACCTTTAAGA CCAATGACTT ACAAGGCAGC TGTAGATCTT AGCCACTTTT 6781 TAAAAGAAAA GGGGGGACTG GAAGGGCTAA TTCACTCCCA ACGAAGACAA GATGGGATCA 6841 ATTCACCATG GGAATAACTT CGTATAGCAT ACATTATACG AAGTTATGCT GCTTTTTGCT 6901 TGTACTGGGT CTCTCTGGTT AGACCAGATC TGAGCCTGGG AGCTCTCTGG CTAACTAGGG 6961 AACCCACTGC TTAAGCCTCA ATAAAGCTTG CCTTGAGTGC TTCAAGTAGT GTGTGCCCGT 7021 CTGTTGTGTG ACTCTGGTAA CTAGAGATCC CTCAGACCCT TTTAGTCAGT GTGGAAAATC 7081 TCTAGCAGCA TCTAGAATTA ATTCCGTGTA TTCTATAGTG TCACCTAAAT CGTATGTGTA 7141 TGATACATAA GGTTATGTAT TAATTGTAGC CGCGTTCTAA CGACAATATG TACAAGCCTA 7201 ATTGTGTAGC ATCTGGCTTA CTGAAGCAGA CCCTATCATC TCTCTCGTAA ACTGCCGTCA 7261 GAGTCGGTTT GGTTGGACGA ACCTTCTGAG TTTCTGGTAA CGCCGTCCCG CACCCGGAAA 7321 TGGTCAGCGA ACCAATCAGC AGGGTCATCG CTAGCCAGAT CCTCTACGCC GGACGCATCG 7381 TGGCCGGCAT CACCGGCGCC ACAGGTGCGG TTGCTGGCGC CTATATCGCC GACATCACCG 7441 ATGGGGAAGA TCGGGCTCGC CACTTCGGGC TCATGAGCGC TTGTTTCGGC GTGGGTATGG 7501 TGGCAGGCCC CGTGGCCGGG GGACTGTTGG GCGCCATCTC CTTGCATGCA CCATTCCTTG 7561 CGGCGGCGGT GCTCAACGGC CTCAACCTAC TACTGGGCTG CTTCCTAATG CAGGAGTCGC 7621 ATAAGGGAGA GCGTCGAATG GTGCACTCTC AGTACAATCT GCTCTGATGC CGCATAGTTA 7681 AGCCAGCCCC GACACCCGCC AACACCCGCT GACGCGCCCT GACGGGCTTG TCTGCTCCCG 7741 GCATCCGCTT ACAGACAAGC TGTGACCGTC TCCGGGAGCT GCATGTGTCA GAGGTTTTCA7801 CCGTCATCAC CGAAACGCGC GAGACGAAAG GGCCTCGTGA TACGCCTATT TTTATAGGTT 7861 AATGTCATGA TAATAATGGT TTCTTAGACG TCAGGTGGCA CTTTTCGGGG AAATGTGCGC 7921 GGAACCCCTA TTTGTTTATT TTTCTAAATA CATTCAAATA TGTATCCGCT CATGAGACAA 7981 TAACCCTGAT AAATGCTTCA ATAATATTGA AAAAGGAAGA GTATGAGTAT TCAACATTTC 8041 CGTGTCGCCC TTATTCCCTT TTTTGCGGCA TTTTGCCTTC CTGTTTTTGC TCACCCAGAA 8101 ACGCTGGTGA AAGTAAAAGA TGCTGAAGAT CAGTTGGGTG CACGAGTGGG TTACATCGAA 8161 CTGGATCTCA ACAGCGGTAA GATCCTTGAG AGTTTTCGCC CCGAAGAACG TTTTCCAATG 8221 ATGAGCACTT TTAAAGTTCT GCTATGTGGC GCGGTATTAT CCCGTATTGA CGCCGGGCAA 8281 GAGCAACTCG GTCGCCGCAT ACACTATTCT CAGAATGACT TGGTTGAGTA CTCACCAGTC 8341 ACAGAAAAGC ATCTTACGGA TGGCATGACA GTAAGAGAAT TATGCAGTGC TGCCATAACC 8401 ATGAGTGATA ACACTGCGGC CAACTTACTT CTGACAACGA TCGGAGGACC GAAGGAGCTA 8461 ACCGCTTTTT TGCACAACAT GGGGGATCAT GTAACTCGCC TTGATCGTTG GGAACCGGAG 8521 CTGAATGAAG CCATACCAAA CGACGAGCGT GACACCACGA TGCCTGTAGC AATGGCAACA 8581 ACGTTGCGCA AACTATTAAC TGGCGAACTA CTTACTCTAG CTTCCCGGCA ACAATTAATA 8641 GACTGGATGG AGGCGGATAA AGTTGCAGGA CCACTTCTGC GCTCGGCCCT TCCGGCTGGC 8701 TGGTTTATTG CTGATAAATC TGGAGCCGGT GAGCGTGGGT CTCGCGGTAT CATTGCAGCA 8761 CTGGGGCCAG ATGGTAAGCC CTCCCGTATC GTAGTTATCT ACACGACGGG GAGTCAGGCA 8821 ACTATGGATG AACGAAATAG ACAGATCGCT GAGATAGGTG CCTCACTGAT TAAGCATTGG 8881 TAACTGTCAG ACCAAGTTTA CTCATATATA CTTTAGATTG ATTTAAAACT TCATTTTTAA 8941 TTTAAAAGGA TCTAGGTGAA GATCCTTTTT GATAATCTCA TGACCAAAAT CCCTTAACGT 9001 GAGTTTTCGT TCCACTGAGC GTCAGACCCC GTAGAAAAGA TCAAAGGATC TTCTTGAGAT 9061 CCTTTTTTTC TGCGCGTAAT CTGCTGCTTG CAAACAAAAA AACCACCGCT ACCAGCGGTG 9121 GTTTGTTTGC CGGATCAAGA GCTACCAACT CTTTTTCCGA AGGTAACTGG CTTCAGCAGA 9181 GCGCAGATAC CAAATACTGT CCTTCTAGTG TAGCCGTAGT TAGGCCACCA CTTCAAGAAC 9241 TCTGTAGCAC CGCCTACATA CCTCGCTCTG CTAATCCTGT TACCAGTGGC TGCTGCCAGT 9301 GGCGATAAGT CGTGTCTTAC CGGGTTGGAC TCAAGACGAT AGTTACCGGA TAAGGCGCAG 9361 CGGTCGGGCT GAACGGGGGG TTCGTGCACA CAGCCCAGCT TGGAGCGAAC GACCTACACC 9421 GAACTGAGAT ACCTACAGCG TGAGCATTGA GAAAGCGCCA CGCTTCCCGA AGGGAGAAAG 9481 GCGGACAGGT ATCCGGTAAG CGGCAGGGTC GGAACAGGAG AGCGCACGAG GGAGCTTCCA 9541 GGGGGAAACG CCTGGTATCT TTATAGTCCT GTCGGGTTTC GCCACCTCTG ACTTGAGCGT 9601 CGATTTTTGT GATGCTCGTC AGGGGGGCGG AGCCTATGGA AAAACGCCAG CAACGCGGCC 9661 TTTTTACGGT TCCTGGCCTT TTGCTGGCCT TTTGCTCACA TGTTCTTTCC TGCGTTATCC 9721 CCTGATTCTG TGGATAACCG TATTACCGCC TTTGAGTGAG CTGATACCGC TCGCCGCAGC 9781 CGAACGACCG AGCGCAGCGA GTCAGTGAGC GAGGAAGCGG AAGAGCGCCC AATACGCAAA 9841 CCGCCTCTCC CCGCGCGTTG GCCGATTCAT TAATGCAGCT GTGGAATGTG TGTCAGTTAG 9901 GGTGTGGAAA GTCCCCAGGC TCCCCAGCAG GCAGAAGTAT GCAAAGCATG CATCTCAATT 9961 AGTCAGCAAC CAGGTGTGGA AAGTCCCCAG GCTCCCCAGC AGGCAGAAGT ATGCAAAGCA 10021 TGCATCTCAA TTAGTCAGCA ACCATAGTCC CGCCCCTAAC TCCGCCCATC CCGCCCCTAA 10081 CTCCGCCCAG TTCCGCCCAT TCTCCGCCCC ATGGCTGACT AATTTTTTTT ATTTATGCAG 10141 AGGCCGAGGC CGCCTCGGCC TCTGAGCTAT TCCAGAAGTA GTGAGGAGGC TTTTTTGGAG 10201 GCCTAGGCTT TTGCAAAAAG CTTGGACACA AGACAGGCTT GCGAGATATG TTTGAGAATA 10261 CCACTTTATC CCGCGTCAGG GAGAGGCAGT GCGTAAAAAG ACGCGGACTC ATGTGAAATA 10321 CTGGTTTTTA GTGCGCCAGA TCTCTATAAT CTCGCGCAAC CTATTTTCCC CTCGAACACT 10381 TTTTAAGCCG TAGATAAACA GGCTGGGACA CTTCACATGA GCGAAAAATA CATCGTCACC10441 TGGGACATGT TGCAGATCCA TGCACGTAAA CTCGCAAGCC GACTGATGCC TTCTGAACAA10501 TGGAAAGGCA TTATTGCCGT AAGCCGTGGC GGTCTGTACC GGGTGCGTTA CTGGCGCGTG10561 AACTGGGTAT TCGTCATGTC GATACCGTTT GTATTTCCAG CTACGATCAC GACAACCAGC10621 GCGAGCTTAA AGTGCTGAAA CGCGCAGAAG GCGATGGCGA AGGCTTCATC GTTATTGATG10681 ACCTGGTGGA TACCGGTGGT ACTGCGGTTG CGATTCGTGA AATGTATCCA AAAGCGCACT10741 TTGTCACCAT CTTCGCAAAA CCGGCTGGTC GTCCGCTGGT TGATGACTAT GTTGTTGATA10801 TCCCGCAAGA TACCTGGATT GAACAGCCGT GGGATATGGG CGTCGTATTC GTCCCGCCAA10861 TCTCCGGTCG CTAATCTTTT CAACGCCTGG CACTGCCGGG CGTTGTTCTT TTTAACTTCA10921 GGCGGGTTAC AATAGTTTCC AGTAAGTATT CTGGAGGCTG CATCCATGAC ACAGGCAAAC10981 CTGAGCGAAA CCCTGTTCAA ACCCCGCTTT AAACATCCTG AAACCTCGAC GCTAGTCCGC11041 CGCTTTAATC ACGGCGCACA ACCGCCTGTG CAGTCGGCCC TTGATGGTAA AACCATCCCT11101 CACTGGTATC GCATGATTAA CCGTCTGATG TGGATCTGGC GCGGCATTGA CCCACGCGAA11161 ATCCTCGACG TCCAGGCACG TATTGTGATG AGCGATGCCG AACGTACCGA CGATGATTTA11221 TACGATACGG TGATTGGCTA CCGTGGCGGC AACTGGATTT ATGAGTGGGC CCCGGATCTT11281 TGTGAAGGAA CCTTACTTCT GTGGTGTGAC ATAATTGGAC AAACTACCTA CAGAGATTTA11341 AAGCTCTAAG GTAAATATAA AATTTTTAAG TGTATAATGT GTTAAACTAC TGATTCTAAT11401 TGTTTGTGTA TTTTAGATTC CAACCTATGG AACTGATGAA TGGGAGCAGT GGTGGAATGC11461 CTTTAATGAG GAAAACCTGT TTTGCTCAGA AGAAATGCCA TCTAGTGATG ATGAGGCTAC11521 TGCTGACTCT CAACATTCTA CTCCTCCAAA AAAGAAGAGA AAGGTAGAAG ACCCCAAGGA11581 CTTTCCTTCA GAATTGCTAA GTTTTTTGAG TCATGCTGTG TTTAGTAATA GAACTCTTGC11641 TTGCTTTGCT ATTTACACCA CAAAGGAAAA AGCTGCACTG CTATACAAGA AAATTATGGA11701 AAAATATTCT GTAACCTTTA TAAGTAGGCA TAACAGTTAT AATCATAACA TACTGTTTTT11761 TCTTACTCCA CACAGGCATA GAGTGTCTGC TATTAATAAC TATGCTCAAA AATTGTGTAC11821 CTTTAGCTTT TTAATTTGTA AAGGGGTTAA TAAGGAATAT TTGATGTATA GTGCCTTGAC11881 TAGAGATCAT AATCAGCCAT ACCACATTTG TAGAGGTTTT ACTTGCTTTA AAAAACCTCC11941 CACACCTCCC CCTGAACCTG AAACATAAAA TGAATGCAAT TGTTGTTGTT AACTTGTTTA12001 TTGCAGCTTA TAATGGTTAC AAATAAAGCA ATAGCATCAC AAATTTCACA AATAAAGCAT12061 TTTTTTCACT GCATTCTAGT TGTGGTTTGT CCAAACTCAT CAATGTATCT TATCATGTCT12121 GGATCAACTG GATAACTCAA GCTAACCAAA ATCATCCCAA ACTTCCCACC CCATACCCTA12181 TTACCACTGC CAATTACCTA GTGGTTTCAT TTACTCTAAA CCTGTGATTC CTCTGAATTA12241 TTTTCATTTT AAAGAAATTG TATTTGTTAA ATATGTACTA CAAACTTAGT//pLOX-Ttag-iresTK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-hOKMS FUW-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-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-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。