lentivirus generation 慢病毒转染手册
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.。
慢病毒转染步骤
转染法步骤：
1分至适宜浓度的细胞到六孔板中，次日细胞应30%~50%融合
2.第二天，吸掉板中的生长培养基并加入新的含聚凝胺的培养基，加入适当数量的病毒于细胞中（见“决定慢病毒效价”部分），每孔溶液终体积为3ml
注意：考虑到细胞在自旋过程中不一定能完全被培养基覆盖，不推崇减少六孔板中的溶液终体积。
调整孔中聚凝胺的终浓度为8mg/ml.通过涡旋六孔板轻轻地混合培养基，病毒以及聚凝胺。
3.为减少自旋-转染过程中悬浮物质的形成以及孔与孔之间液体接触的情况发生，用无菌微孔密封膜封闭六孔板（直接盖在板上），封闭好后，再在密封膜上加上一层未经灭菌的密封箔，最后盖上六孔板盖。
4.在标准的组织培养离心机中，37°C，2250rpm（1200g）条件下自旋-转染细胞60min.
5.一自旋-转染后就移走密封膜和密封箔，将孔中原有的培养基换为新鲜的生长培养基（每孔2ml就足够）
6.转染24h后，吸掉孔中的培养基并每孔加入新鲜的含有嘌呤霉素的2ml生长培养基。
注意：嘌呤霉素的浓度应被每个细胞株充分利用，一般规定在1-5μg/mL
7.进一步的培养时间主要取决于细胞株的类型以及转染后的实验分析。
通常，嘌呤霉素筛选细胞需要大约48h。
下面推荐的是结合给定的细胞株和分析实验来得到的嘌呤霉素筛选
时间
>转染后实验分析嘌呤霉素筛选细胞时间> mRNA knockdown (qPCR) 2+ days
> Protein knockdown (Western) 3+ days
> Phenotypic assay 表型分析3+ days
8.检测目的感染细胞的表面分子。
慢病毒使用手册
慢病毒使用手册慢病毒（Lentivirus）是一类非常常见的病毒，它属于逆转录病毒家族。
与其他病毒相比，慢病毒具有一些特殊的特征，使其在生物研究领域和基因治疗等应用中变得非常重要。
本文将介绍慢病毒的基本特征、制备和使用方法，以及慢病毒在基因转染、基因表达和基因治疗中的应用。
一、慢病毒的基本特征慢病毒是一类具有外包膜的病毒，其基因组由一条单链正义RNA组成。
慢病毒具有较大的基因载量能力，可携带长达9kb的外源DNA序列。
另外，慢病毒具有高度的细胞性选择性，能够感染多种哺乳动物细胞，并将外源基因稳定地集成到细胞基因组中。
二、慢病毒的制备方法慢病毒的制备包括构建慢病毒载体和包装慢病毒。
构建慢病毒载体通常采用三元质粒系统，其中包括基因载体、包装载体和包衣载体。
基因载体负责携带外源基因序列，而包装载体负责表达与慢病毒复制有关的基因，如gag、pol和env等。
包衣载体则负责表达包衣蛋白，使慢病毒能够正常装配和释放。
包装慢病毒的方法通常采用转染细胞的方式。
将构建好的慢病毒载体与包装载体和包衣载体一同转染到特定的细胞中，通过包装载体表达的基因来启动慢病毒的复制和包装过程。
经过适当的培养和处理后，可以获得高效包装的慢病毒颗粒。
三、慢病毒的使用方法慢病毒主要通过基因转染、基因表达和基因治疗等方式应用于生物研究和医学领域。
1. 基因转染：慢病毒可以用于将外源基因导入到细胞中，实现基因转染。
通过选择性的感染特定细胞或细胞系，可以研究和探索特定基因的功能和调控机制。
2. 基因表达：慢病毒可以被用作基因表达工具。
外源基因在细胞内被稳定地整合到基因组中，从而实现长期稳定的基因表达。
慢病毒可以用于产生稳定的细胞株，并通过基因敲入或敲除等方法研究基因功能。
3. 基因治疗：慢病毒在基因治疗中的应用非常广泛。
通过将修正后的基因导入到患者体内的细胞中，可以实现对某些慢病毒引起的遗传疾病的基因治疗。
此外，慢病毒载体还可以用于制备疫苗和用于癌症免疫治疗等领域。
原汁原味的慢病毒转染与感染protocol
原汁原味的慢病毒转染与感染protocol Lentivirustransfection and infectionsPackaging cell line:293FTGrowth medium:DMEM containing 10% HI FBS, 1%P/S, 0.5mg/ml G418,4 mM L-Glutamine,0.1 mM MEM Non-Essential Amino Acids,1 mM MEM sodium pyruvateLentiviral medium:DMEM containing 10% HI FBS, without antibiotics,4 mML-Glutamine, 0.1 mM MEM Non-Essential Amino Acids,1 mM MEM sodium pyruvateTargetcell medium:DMEM or other medium containing 10% HIFBS, without antibiotics.ProtocolCulturing 293FT cellsQuickly thaw the frozen 293FT cells, and immediately after thawing, transfer the cells to a 15 ml tube containing 10 ml PBS,and then pellet the cells. Resuspend the cells in growth medium, and incubat e cells at 37°C in a humidified 5% CO2 incubator.Transfection and infections1. On Day 1, prepare DNA-Lipofectamine? 2000 complexes foreach sample.a.In a sterile 5 ml tube, dilute 3 μg pLP1, 3 μg pLP2, 3 μg pLP/VSVG, and 3 μg of pLenti expression plasmid DNA (12μg total) in 1.5 ml of DMEM (noserum, no P/S). Mix gently.b.In a separate sterile 5 ml tube, dilute 36 μl Lipofectamine? 2000 (mix gently before use) in 1.5 ml of DMEM (no serum, no P/S). Mix gently and incubate for 5 minutes at room temperature.c.After incubation, combine the diluted DNA (Step a) with the diluted Lipofectamine? 2000 (Step b). Mix gently.d.Incubate for 20 minutes at room temperature to allow the DNA Lipofectamine ? 2000 complexes to form. The solution may appear cloudy, but this will not impact the transfection.e.While DNA-lipid complexes are forming, trypsinize and count the 293FT cells.Resuspend the cells at a density of 1.2x 106 cells/ml in lentiviral medium.f.Add the DNA-Lipofectamine? 2000 complexes (Step 1d) to a 10 cm tissue cultureplate containing 5 ml of lentiviral medium.g. Add 5 ml of the 293FT cell suspension from Step 2 (6 x 106 totalcells) to the plate containing media and DNA-Lipofectamine? 2000 complexes (Step 3).Mix gently by rocking the plate back and forth. Incubate cells overnight at 37°Cin a humidified 5% CO2 incubator.2.The next day (Day 2),remove and discard the medium containing the DNA Lipofectamine ? 2000 complexes and replace with 10 ml lentiviral medium.Incubate cells for 48 hours at 37°C in a humi dified 5% CO2 incubator.Note:Expression of the VSV G glyco protein causes293FT cells to fuse, resulting in the appearance of large, multinucleated cells known as syncytia. This morphological changeis normal and does not affect production of the lentivirus.3. Day 3, set up the targetcell line in target cell medium to 60mm plate so that they will be 30% confluent on the next day.4. Posttransfection (Day 4),a. harvestvirus-containing supernatants. Use a 10 ml syringe to remove the medium from the 293FT cell lines, and filter the viral supernatants through a 0.45 μm filter in 15 ml sterile tube. Replace the medium removed from the packaging cells with 10 ml lentiviral medium.b. infect target cells: 1 volume of lentiviral medium (2ml) and1 volume offiltered virus-containing supernatants(2ml), with polybrene 4 μg/ml.c.Pipet the retaining viral supernatants into 1.5 ml sterile tube in 1 ml aliquots. Store viral stocks at -80°C. When stored properly, viral stocks ofan appropriate titer should be suitable for use for up to one year. When using the frozen viral stocks, thaw frozen stocksat room temperature, rather than at 37°C, since lentivirus is temperature-sensitive.5. Day 5 or 6, second round of infection: repeat the infection as described on day 4. And throw the 293FT cells.6.Day 6 or 7, split target cells to 100 mm plate with fresh target growth medium.7. On the Day 7or 8, select cells using drug.。
慢病毒感染细胞 操作手册
慢病毒感染细胞实验方法一、实验概述培养生长状态良好的目的细胞，根据慢病毒感染预实验结果设计各组实验条件，进行正式感染。
若为荧光标记的慢病毒感染，参照预实验确定的感染时间点，于荧光显微镜下观察GFP表达情况，荧光率达70-80％左右，细胞汇合度达80%左右，收集细胞进入下游实验。
若为抗性基因（如Puromycin）标记的慢病毒感染，感染48 h-72h后，使用抗生素筛选48h，收集细胞汇合度70%-80%左右的生长状态良好的细胞进行下游实验。
二、实验材料1.主要试剂试剂名称试剂来源cat.No.胎牛血清Ausbian VS500TDMEM Corning 10-013-CVR胰酶生工生物工程（上海）股份有限公司T0458-50G Puromycin Clontech 631305D-Hanks 上海吉凯基因技术有限公司配制2.主要仪器仪器名称仪器来源cat.No.荧光显微镜奥林帕斯IX71CO2培养箱日本三洋SANYO MCO-175倒置显微镜上海蔡康光学仪器有限公司XDS-100离心机赛默飞世尔科技(中国)有限公司Fresco 21生物安全柜上海振样创空气净化设备有限公司Bio 1200-Ⅱ-A2三、实验步骤1．准备目的细胞（1）从液氮罐中取出细胞冻存管，迅速放入37℃水浴中，并不时摇动使其尽快解冻。
（2）完全解冻后，1300 rpm，离心3 min，75%酒精擦拭冻存管消毒后，移至生物安全柜。
（3）吸去冻存液上清，加入1 mL新鲜的完全培养基重悬细胞，将细胞悬液接种至含有3 mL完全培养基的6-cm dish 中，轻轻晃匀后置于37℃、5% CO2培养箱。
（4）24 h后更换一次培养液继续培养，待细胞汇合度达80%左右传代培养，保持细胞良好的生长状态。
2．目的细胞慢病毒感染（1）贴壁细胞：a.处于对数生长期的细胞胰酶消化，完全培养基制成3-5×104个/mL细胞悬液，并根据表1接种相应的细胞数到培养板中，继续培养保证感染时铺板量达到15-30%左右。
慢病毒专用转染试剂说明书
慢病毒专用转染试剂LentiFit TM使用说明书产品简介：LentiFit TM脂质体转染试剂是一种高效的阳离子脂质体转染试剂。
与其他脂质体转染试剂相比，汉恒生物独立研发的LentiFit TM脂质体转染试剂具有转染效率高，重复性好，操作简单，无明显细胞毒性，抗血清干扰等优点。
对于大多数细胞，用LentiFit TM转染后72小时内不更换培养液无明显细胞毒性。
同时，血清的存在不影响LentiFit TM的转染效率，这样可以减小无血清对细胞的损伤。
基于以上优点，LentiFit TM常用做慢病毒包装的转染试剂。
包装清单以及储存条件：试剂名货号储存条件规格有效期LentiFit TM转染试剂Let10004℃保存 1 mL12个月使用方法：1.细胞培养：以1个10 cm培养皿转染293T细胞为例，转染前一天接种细胞(20-24小时)，接种细胞约3.5×106，接种体积10 mL，确保第二天转染时细胞汇合度（confluent）能达到约50%~70%。
2.在进行转染步骤前，把10 cm培养皿中的培养液更换成新鲜细胞培养液，培养液的体积为5 mL，放回培养箱中继续培养，并准备转染体系（步骤3-6）。
3. 把LentiFit TM脂质体转染试剂轻轻混匀。
4. 对于待转染的10 cm培养皿中的细胞，取一只洁净无菌离心管，加入24 µg质粒（目的质粒和辅助质粒的比例根据不同包装系统进行调整）到500 µL DMEM溶液，用枪轻轻吹打混匀。
5. 取另一只洁净无菌离心管，加入500 µL DMEM溶液，再加入40 µL LentiFit TM，用枪轻轻吹打混匀,室温放置5分钟。
6. 将步骤4与5中的质粒溶液与LentiFit TM溶液混合，用枪轻轻吹打混匀。
不可V ortex 或离心。
室温孵育20分钟。
有可能出现絮状沉淀物，属正常现象，不会影响转染效率。
7. 将转染复合物逐滴加入到培养皿中，轻轻“8”字形摇晃混匀后，放回培养箱继续培养。
贴壁细胞慢病毒转染步骤及方法
贴壁细胞慢病毒转染步骤及方法
一、细胞培养Panc-1 细胞，常规培养使用含10% FBS (GIBCO) 的DMEM 培养基（含1.5 mg/L-Glutamine，100 U/mL penicillin，100 μg/mL Streptomycin) 中，37ºC 5% CO2饱和湿度培养箱中培养。
二、病毒侵染实验材料及试剂DMEM培养基+ 10% FBSPBS（Life Science Products&Services）Trypsin-EDTA Solution (Gibco)24孔板（Corning）Lentivirus-NC 病毒液（GenePharma，1×109 TU/mL）
三、步骤
1. 将状态良好的Panc-1 消化后重悬，取适量细胞接种至24 孔板中，37°C培养箱中过夜；
2. 取阴性对照病毒，按1:10、1:100、1:1000 与DMEM培养基混合稀释，总体积约500 μL，并加入终浓度5 μg/mL Polybrene；
3. 吸去24 孔板中原培养基，换入阴性对照病毒梯度稀释液，37°C培养箱中培养；
4. 24h 后吸去阴性对照病毒稀释液，换入500 μL新鲜培养基，37°C培养箱中培养；
5. 48-96h 于荧光倒置显微镜下观察并记录结果。
悬浮细胞慢病毒转染步骤及方法
悬浮细胞慢病毒转染步骤及方法
实验材料与方法
一、细胞培养人红白血病细胞Kasumi-1，常规培养使用含10% FBS(GIBCO)的RPMI 1640 培养基（Hyclone）（含1.5 mg/L-Glutamine，100 U/mL Penicillin，100 μg/mL Streptomycin) 中，37ºC 5% CO2饱和湿度培养箱中培养。
二、病毒侵染实验材料及试剂
DMEM培养基+ 10% FBSRPMI 1640培养基+10% FBSD-Hank’s Solution96孔板（Corning）24孔板（Corning）Lentivirus-病毒液（GenePharma，1×108 TU/mL）步骤1. 稀释病毒：稀释液（靶细胞维持液培养基）400 μL + 终浓度5 μg/mL Polybrene，将慢病毒原液按1:20、1:10、1:5 加入到稀释液中；
2. 24-well，按4×105 cells/well（根据细胞种类调整），1000 RPM 3min，取细胞沉淀。
将Step 1 中各份病毒稀释液分别与每份细胞沉淀重悬，同时建立对照（blank、negative），37°C 5% CO2中培养；
3. 12~24h 后离心移去细胞侵染后的病毒液，加入0.5 mL 完全培液，37°C 5% CO2过夜
4. 根据细胞状态和类型，如果必要分出1/3~1/5，加入0.5 mL完全培养，继续培养24~48h；荧光倒置显微镜下观察结果。
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Generation o f L entivirus
Reagents
•293T: ATCC
•Fugene 6 transfection reagent: Roche cat# 11814443001
•FBS: Invitrogen cat# 16000-044
•DMEM: Invitrogen cat# 11965-118
•Pen/strep: Invitrogen cat# 15140-155
•L-glutamine: Invitrogen cat# 25030-156
•Non essential amino acid (NEAA): Invitrogen cat# 11140-050
•Amicon Ultra Ultracel 100K: Millipore: cat# UFC910024
•Beckman centrifuge tubes: Beckman: cat# 344058
293T/fibroblast media (500 mls):
DMEM (450 ml)
10% FBS (50 ml)
Pen/strep (5ml)
L-glutamine (5ml)
NEAA (5 ml)
Reagent setup
Lentiviral vectors:Prepare all vector plasmids with Qiagen Maxiprep kits, including lentiviral packaging vectors and lentiviral transfer vectors
A. Production of Virus
1. Seed 2x106 293T cells on a 100-cm tissue culture dish and incubate overnight until cells
reach ~70% confluence (~1-2 days). Replace with 10 ml of fresh media 2 hours before transfection.
2. Mix lentiviral transfer vector and packaging vectors in 600 ul of DMEM in an eppendorf
tube. Add 50 ul of Fugene6 directly to the DNA mixture, making sure Fugene6 does not come in contact with the plastic. Gently vortex tube containing transfection mixture and incubate at RT for 20 min.
Second generation packaging system
Transfer vector 10 ug
pMD2.G 5 ug
psPAX2 5 ug
Third generation packaging system
Transfer vector 10 ug
pMDL g/pRRE 5 ug
pRSV-Rev 2.5 ug
pMD2.G 2.5 ug
3. Add transfection mixture dropwise to cells; incubate 4 hrs to overnight (16 hours) and
replace with fresh medium.
4. Collect virus-containing medium 48 hours after transfection and replace with fresh
medium. Collect virus every 12 hours for up to 3 times total. Keep all viral media at 4C
until all collections are done. Pass viral media through a 0.45uM low protein-binding filter.
At this point, the viral supernatant can be used to infect cells, frozen at -80C or
concentrated.
Concentration using Amicon Ultra Centrifugal Filters:
Transfer viral supernatnat to Amicon Filter and spin filter in tabletop centrifuge at 3000 rpm for 10-20 min at 4C. Concentrated virus can be aliquoted and stored at -80C. Thaw an
aliquot on ice before use; do not refreeze.
Concentration by ultracentrifugation:
1. Transfer viral supernatant to 33ml Beckman ultracentrifugation tubes and spin for 2
hrs at 24,000 rpm using SW32Ti or SW28Ti.
2. Pour off supernatant carefully and resuspend viral pellet using 100-ul pipette tip with
appropriate amount of DMEM to concentrate virus 100x. It may take 30 min to
overnight at 4C for the pellet to completely dissolve.
3. Store virus in aliquots at -80C. Thaw an aliquot on ice before each use; do not
refreeze.。