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May 2015

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Generation of Stable Expression Mammalian Cell Lines Using Lentivirus
利用慢病毒制备稳定表达哺乳动物细胞系   

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Abstract

Lentiviruses are used very widely to generate stable expression mammalian cell lines. They are used for both gene down-regulation (by using shRNA) or for gene up-regulation (by using ORF of gene of interest). The technique of generating stable cell lines using 3rd generation lentivirus is very robust and it typically takes about 1-2 weeks to get stable expression for most mammalian cell lines. The advantage of using the 3rd generation lentivirus are that are very safe and they are replication incompetent.

Keywords: Lentivirus (慢病毒), Stable cell lines (稳定细胞系), Gene knockdown (基因敲低), Gene over-expression (基因过表达), Mammalian cell lines (哺乳动物细胞系), Transduction (转染)

Background

As compared to the short term protein expression resulting from transient transfection, stable cell lines generated using lentivirus exhibit long term protein expression and the system is highly reproducible (LaGory et al., 2015). Below is the outline for generation of stable cell lines using lentivirus (Figure 1). In this protocol, we describe the use of the 3rd generation lentiviral system which uses three different plasmids for generating stable cell lines. First plasmid contains your gene of interest usually flanked by Long Terminal Repeat (LTR) sequences, that are integrated into the host genome. A variety of these plasmids can be obtained from Addgene. Second is the pCMV Delta R8.2 plasmid that encodes all components necessary for packaging the lentivirus viz. HIV-1 Gag, Pol, Tat and Rev. Third and the final plasmid is the pCMV VSVG that encodes the viral envelope protein.


Figure 1. Schematic representation of generation of stable cell lines using lentivirus

Materials and Reagents

  1. 10 cm2 tissue culture treated plates (Corning, catalog number: CLS430165)
  2. Low protein binding 0.45 μm PDVF filters (Millipore, catalog number: SLHV033RS)
  3. 10 ml plastic syringes sterile (any brand)
  4. Sterile Glass jar
  5. Disposable plastic pipettes (5 ml and 10 ml)
  6. Sterile 1.5 ml Eppendorf tubes
  7. Pasteur pipettes
  8. HEK293T (293T) (ATCC, catalog number: CRL-3216)
  9. Target cell line (any human cancer cell line)
  10. Xtreme gene 9 transfection reagent (Sigma-Aldrich, 636577900)
  11. Packaging plasmids: pCMV delta R8.2 (Addgene, catalog number: 12263) and pCMV-VSV-G (Addgene, catalog number: 8454) and your plasmid of interest (test and control plasmid – see Notes)
  12. 10% bleach
  13. Polybrene solution: 8 mg/ml in water filter sterilized (Hexadimethrine bromide, Sigma, catalog number: H9268), made fresh every time 
  14. Opti-MEM reduced serum medium (Thermo Fisher, catalog number: 31985062)
  15. DMEM complete medium with 10% FBS and pen-strep
  16. Selection antibiotic (e.g., Puromycin)
  17. Dry ice or liquid nitrogen
  18. DMSO
  19. Liquid nitrogen

Equipment

  1. Cell culture incubators (37 °C, 5% CO2 and 32 °C, 5% CO2)
  2. Hemocytometer (Standard)
  3. Laminar hood BSL-2 type
  4. 37 °C water bath
  5. Autoclave
  6. Micropipettes (1,000 μl, 200 μl)
  7. -80 °C freezer

Procedure

  1. Preparation of lentivirus in HEK293T cells
    1. Day 0: Seed about 2-3 million HEK293T cells in a 10 cm2 plate.
    2. Day 1: HEK293T cells should be about 70-80% confluent. If for some reason they seem less you may wait for another
      8-10 h. 
    3. Prepare transfection mix using the following proportions in a sterile 1.5 ml Eppendorf tube as per manufacturer’s instructions:
      Opti-MEM medium
      1 ml
      pCMV delta R8.2
      2 μg
      pCMV-VSV-G
      0.5 μg
      Target plasmid
      1.5 μg
      Xtreme gene 9
      12 μl (1:3, DNA:Xtreme Gene, Ratio)
      Once you add all the five components in a 1.5 ml Eppendorf tube, mix the solution by pipetting or inverting. Do not vortex. Incubate the mix for about 20 min at room temperature.
    4. Aspirate the medium from HEK293T cells using Pasteur pipettes and add about 9 ml fresh complete DMEM medium.
    5. After 20 min incubation of the transfection mix, add the mix to the HEK293T using a 200 μl micropipette. Make sure to add the mix drop-by-drop to cover the complete area of the plate. 
    6. Incubate the plate in a 37 °C cell culture incubator for about 48 h.
    7. Day 3: Using a 10 ml disposable sterile pipette, collect the supernatant/media off the HEK293T plates in a sterile 15 ml tube. Add another fresh 10 ml DMEM media to the plate and move it back to the incubator. Transfer the collected supernatant into a 10 ml syringe and then filter it through a 0.45 μm PVDF filter inside the laminar hood. This step will result in collection of filtered viral supernatant without any residual HEK293T cells in it.
      Note: To ensure proper disposal of all the equipment that has come in contact with the virus, all the pipettes, tips, syringes and filters used here must be washed with 10% bleach for about 10 min before finally discarding them in a biohazard bin. 
    8. If you do not have your target cells ready or if you plan to use the lentivirus later, you can snap freeze the viral supernatant using dry ice or liquid nitrogen and then store it at -80 °C. The virus is usually stable for several months at this temperature.
    9. Day 4: If desired, another round of 10 ml lentiviral supernatant can be collected from still growing HEK293T cell plates, similar to Day 3. Before discarding the HEK293T cell plates in the biohazard bin, they should be treated with 10% bleach for about 5-10 min. 
    Pro-tip: The probability of high viral titer can be estimated from the amount of HEK293T cell death, i.e., the more the cell death, the more is the probability of collection of virus of high titer. 

  2. Preparation of target cells
    1. Day 2 (if you want to do this in parallel with the virus preparation, Recommended): Seed about 0.5-1 x 106 target cells each in two 10 cm2 tissue culture plates. One plate is where you will add the viral supernatant, i.e., the lentivirus and the other will be your killing control where you will not add any lentivirus.
    2. Day 3: The target cells should be around 40%-50% confluent prior to transduction, i.e., addition of virus. If your lentivirus is in the freezer, make sure you thaw it in a 37 °C water bath before adding it to the cells. Once the cells are ready, prepare a polybrene medium (target cell medium) mix. The final concentration of polybrene should be 8 μg/ml. Prepare 10x polybrene medium mix by adding 2 ml medium with 20 μl of 8 mg/ml Polybrene. Aspirate the medium in the target cells using Pasteur pipette and add 1 ml polybrene medium mix and let it incubate for about 1-2 min.
      Note: It’s recommended that polybrene solution is made fresh each time OR you can store aliquots of it at -20 °C. Thaw and use an aliquot each time. Do not re-freeze. 
    3. Using a disposable 10 ml pipette, add 9 ml lentivirus to the target cells. Add plain medium to the killing control plate.
    4. Day 4: Repeat the same steps as Day 3, to do a second round of transduction to ensure all the cells get the lentivirus. See the note in Step A7. 
    5. Day 5: Add the selection antibiotic at the killing concentration (e.g., Puromycin 1-2 μg/ml) to both plates. See Notes to determine the killing concentration of the selection antibiotic. Typically, fresh media with antibiotic is added every two days until all the cells in the killing control plate are dead.
    6. Days 8-10: Before you do any further experiments, make sure to cryo-freeze about 1/4th portion of cells in 10% DMSO with DMEM in liquid nitrogen. Passage the rest of the cells for RNA/protein isolation, in order to validate for stable expression of protein of interest.

Notes

  1. Generation of kill curve: Prior to setting out to generate stable cells using lentivirus with a selection maker in any cell line, it is always a good idea to generate a kill curve. A quick google search would tell you a rough range of the concentration of antibiotic to be used. Generally, at least 3-4 different concentrations are tested to ensure complete killing. For example, for puromycin the following concentrations would be tested 1 μg/ml, 2 μg/ml, 3 μg/ml and 4 μg/ml for 48-72 h. 
  2. Test and control plasmid: A proper control plasmid should be used along with the test plasmid. For example, if doing an shRNA-based knockdown then you should include shControl plasmid that has non-targeting sequence instead of the shRNA or if you ORF-based over-expression that the empty vector should be used as a control.

Acknowledgments

This work was supported by NIH Grants CA-67166, CA-198291 and CA-197713, the Sydney Frank Foundation and the Kimmelman Fund (AJG).

Competing interests

The authors declare no conflicts of interest or competing interests.

References

  1. LaGory, E. L., Wu, C., Taniguchi, C. M., Ding, C. K., Chi, J. T., von Eyben, R., Scott, D. A., Richardson, A. D. and Giaccia, A. J. (2015). Suppression of PGC-1α is critical for reprogramming oxidative metabolism in renal cell carcinoma. Cell Rep 12:116-127.

简介

慢病毒非常广泛地用于产生稳定表达的哺乳动物细胞系。 它们用于基因下调(通过使用shRNA)或用于基因上调(通过使用目的基因的ORF)。 使用3 rd 代慢病毒产生稳定细胞系的技术非常稳健,并且通常需要约1-2周才能获得大多数哺乳动物细胞系的稳定表达。 使用3 rd 代慢病毒的优点是非常安全,并且它们是复制不能胜任的。

【背景】 与由瞬时转染产生的短期蛋白质表达相比,使用慢病毒产生的稳定细胞系表现出长期蛋白质表达,并且该系统具有高度可重复性(LaGory 等人,2015)。下面是使用慢病毒产生稳定细胞系的概述(图1)。在该方案中,我们描述了3 rd 代慢病毒系统的使用,该系统使用三种不同的质粒来产生稳定的细胞系。第一个质粒含有您感兴趣的基因,通常侧翼为长末端重复(LTR)序列,它们整合到宿主基因组中。可以从Addgene获得多种这些质粒。第二个是pCMV Delta R8.2质粒,它编码包装慢病毒所需的所有组分。 HIV-1 Gag,Pol,Tat和Rev.第三和最终质粒是编码病毒包膜蛋白的pCMV VSVG。


图1.使用慢病毒生成稳定细胞系的示意图

关键字:慢病毒, 稳定细胞系, 基因敲低, 基因过表达, 哺乳动物细胞系, 转染

材料和试剂

  1. 10cm 2 组织培养处理的平板(Corning,目录号:CLS430165)
  2. 低蛋白结合0.45μmPDVF过滤器(Millipore,目录号:SLHV033RS)
  3. 10毫升塑料注射器无菌(任何品牌)
  4. 无菌玻璃罐
  5. 一次性塑料移液器(5毫升和10毫升)
  6. 无菌1.5毫升Eppendorf管
  7. 巴斯德吸管
  8. HEK293T(293T)(ATCC,目录号:CRL-3216)
  9. 靶细胞系(任何人类癌细胞系)
  10. Xtreme基因9转染试剂(Sigma-Aldrich,636577900)
  11. 包装质粒:pCMV delta R8.2(Addgene,目录号:12263)和pCMV-VSV-G(Addgene,目录号:8454)和您感兴趣的质粒(test and control plasmid - 参见注释)
  12. 10%漂白剂
  13. Polybrene溶液:8 mg / ml水过滤灭菌(Hexadimethrine bromide,Sigma,目录号:H9268),每次都是新鲜的。
  14. Opti-MEM减少血清培养基(Thermo Fisher,目录号:31985062)
  15. DMEM完全培养基,含10%FBS和笔链
  16. 选择抗生素(例如,嘌呤霉素)
  17. 干冰或液氮
  18. DMSO
  19. 液氮

设备

  1. 细胞培养箱(37°C,5%CO 2 和32°C,5%CO 2 )
  2. 血细胞计数器(标准)
  3. 层流罩BSL-2型
  4. 37°C水浴
  5. 高压灭菌器
  6. 微量移液管(1000μl,200μl)
  7. -80°C冰柜

程序

  1. HEK293T细胞中慢病毒的制备
    1. 第0天:在10cm 2 平板中接种约2-3百万个HEK293T细胞。
    2. 第1天: HEK293T细胞应该约70-80%融合。如果由于某种原因他们似乎少了你可能会等待另一个
      8-10小时。 
    3. 按照制造商的说明,使用以下比例在无菌1.5 ml Eppendorf管中制备转染混合物:
      class =“ke-zeroborder”bordercolor =“#000000”style =“width:500px;” border =“0”cellspacing =“0”cellpadding =“2”>Opti-MEM媒体
      1毫升
      pCMV delta R8.2
      2微克
      pCMV-VSV-G
      0.5μg
      靶质粒
      1.5μg
      Xtreme基因9
      12μl(1:3,DNA:Xtreme Gene,Ratio) 将所有五种成分加入1.5 ml Eppendorf管中后,通过移液或倒置混合溶液。不要涡旋。在室温下孵育混合物约20分钟。
    4. 使用巴斯德吸管从HEK293T细胞中吸出培养基,并加入约9ml新鲜的完全DMEM培养基。
    5. 将转染混合物温育20分钟后,使用200μl微量移液管将混合物加入HEK293T中。确保逐滴添加混合物以覆盖板的整个区域。 
    6. 将板在37℃细胞培养箱中孵育约48小时。
    7. 第3天:使用10 ml一次性无菌移液管,从无菌15 ml管中的HEK293T平板上收集上清液/培养基。将另一种新鲜的10ml DMEM培养基加入培养板中,然后将其移回培养箱。将收集的上清液转移到10ml注射器中,然后通过层流罩内的0.45μmPVDF过滤器过滤。该步骤将导致收集过滤的病毒上清液,其中没有任何残留的HEK293T细胞。
      注意:为确保正确处理与病毒接触的所有设备,此处使用的所有移液器,吸头,注射器和过滤器必须用10%漂白剂清洗约10分钟,然后再将其丢弃。生物危害垃圾箱。 
    8. 如果您没有准备好目标细胞或者您打算稍后使用慢病毒,您可以使用干冰或液氮快速冷冻病毒上清液,然后将其储存在-80°C。在这个温度下,病毒通常可以稳定几个月。
    9. 第4天:如果需要,可以从仍然生长的HEK293T细胞板收集另一轮10 ml慢病毒上清液,类似于第3天。在丢弃生物危害箱中的HEK293T细胞板之前,应该对它们进行处理用10%漂白剂约5-10分钟。 
    Pro-tip:高病毒滴度的概率可以从HEK293T细胞死亡的数量来估计,即细胞死亡越多,收集高滴度病毒的概率就越大。 
  2. 靶细胞的制备
    1. 第2天(如果你想与病毒制剂同时进行,推荐):种子约0.5-1 x 10 6 靶细胞每个在两个10cm 2 组织培养板中。一个平板是你将添加病毒上清液的地方,即,慢病毒和另一个将是你的杀戮控制,你不会添加任何慢病毒。
    2. 第3天:在转导之前,靶细胞应该是约40%-50%汇合,即,添加病毒。如果您的慢病毒在冰箱中,请确保在37°C水浴中解冻,然后再将其加入细胞。一旦细胞准备好,准备聚凝胺培养基(靶细胞培养基)混合物。聚凝胺的最终浓度应为8μg/ ml。通过添加2ml含有20μl8mg/ ml Polybrene的培养基制备10x聚凝胺培养基混合物。使用巴斯德吸管吸取目标细胞中的培养基,加入1 ml聚凝胺培养基混合物,孵育约1-2分钟。
      注意:建议每次将聚凝胺溶液制成新鲜的,或者将其等分试样保存在-20°C。每次解冻并使用等分试样。不要重新冻结。 
    3. 使用一次性10 ml移液器,向靶细胞中加入9 ml慢病毒。在杀灭控制板上加入普通培养基。
    4. 第4天:重复与第3天相同的步骤,进行第二轮转导以确保所有细胞都获得慢病毒。请参阅步骤A7中的注释。 
    5. 第5天:将选择的抗生素以杀灭浓度(例如,嘌呤霉素1-2μg/ ml)加入两个平板中。参见注释以确定选择抗生素的杀灭浓度。通常,每两天添加含有抗生素的新鲜培养基,直到杀死对照板中的所有细胞都死亡。
    6. 第8-10天:在进行任何进一步的实验之前,确保在液氮中用DMEM冷冻约10%DMSO中的1/4部分细胞。传代其余细胞进行RNA /蛋白质分离,以验证目的蛋白质的稳定表达。

笔记

  1. 杀灭曲线的产生:在使用任何细胞系中的选择制造者使用慢病毒生成稳定细胞之前,生成杀死曲线总是一个好主意。快速谷歌搜索会告诉你使用抗生素浓度的大致范围。通常,测试至少3-4种不同浓度以确保完全杀死。例如,对于嘌呤霉素,将以1μg/ ml,2μg/ ml,3μg/ ml和4μg/ ml测试以下浓度48-72小时。 
  2. 测试和控制质粒:应与测试质粒一起使用合适的对照质粒。例如,如果进行基于shRNA的敲除,那么你应该包括具有非靶向序列而不是shRNA的shControl质粒,或者如果你基于ORF的过表达,那么空载体应该用作对照。

致谢

这项工作得到NIH Grants CA-67166,CA-198291和CA-197713,悉尼弗兰克基金会和Kimmelman基金(AJG)的支持。

利益争夺

作者声明没有利益冲突或竞争利益。

参考

  1. LaGory,E.L.,Wu,C.,Taniguchi,C.M.,Ding,C.K.,Chi,J.T。,von Eyben,R.,Scott,D.A.,Richardson,A.D。和Giaccia,A.J。(2015)。 抑制PGC-1α对肾细胞癌中氧化代谢的重编程至关重要。 em> Cell Rep 12:116-127。
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引用:Tandon, N., Thakkar, K. N., LaGory, E. L., Liu, Y. and Giaccia, A. J. (2018). Generation of Stable Expression Mammalian Cell Lines Using Lentivirus. Bio-protocol 8(21): e3073. DOI: 10.21769/BioProtoc.3073.
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立奇 殷
18340869839
Without resistance gene screening, when to use fluorescence microscopy to see transfection efficiency
2021/6/18 1:03:05 回复
磊 周
西南医院

Generally, 48 hours after lentivirus infection is enough to observe bright fluorescence under microscope or using flow cytometry.

2021/6/20 21:02:40 回复


Yu Liu
Department of Biology and Biochemistry, University of Houston, the United States of America

For plasmid transfection into 293T cells, 48 hours will give bright fluorescence. For lentiviral transduction into target cells, I usually wait for 72hrs before checking for efficiency.

2021/6/25 13:51:34 回复