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Dec 2020
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Lentivirus-mediated Conditional Gene Expression
慢病毒介导的条件基因表达   

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Abstract

The ability to identify the role of a particular gene within a system is dependent on control of the expression of that gene. In this protocol, we describe a method for stable, conditional expression of Nod-Like receptors (NLRs) in THP-1 cells using a lentiviral expression system. This system combines all the necessary components for tetracycline-inducible gene expression in a single lentivector with constitutive co-expression of a selection marker, which is an efficient means for controlling gene expression using a single viral infection of cells. This is done in a third generation lentiviral expression platform that improves the safety of lentiviruses and allows for greater gene expression than previous lentiviral platforms. The lentiviral expression plasmid is first engineered to contain the gene of interest driven by a TRE (tetracycline response element) promoter in a simple gateway cloning step and is then co-transfected into HEK293T cells, along with packaging and envelope plasmids to generate the virus. The virus is used to infect a cell type of interest at a low MOI so that the majority of the transduced cells contain a single viral integration. Infected cells are grown under selection, and viral integration is validated by qPCR. Gene expression in stably transduced cells is induced with doxycycline and validated by qPCR, immunoblot, and flow cytometry. This flexible lentiviral expression platform may be used for stable and robust induction of a gene of interest in a range of cells for multiple applications.


Graphic abstract:


Schematic overview of lentiviral transduction of THP-1 cells.


Keywords: NLR (NLR), NOD1 (NOD1), Lentivirus (慢病毒 ), Stable expression (稳定表达 ), TRE (TRE), Tetracycline (四环素)

Background

A crucial technique for studying functional responses in molecular biology is the ability to regulate the expression of a gene. Constitutive gene expression systems have allowed researchers to explore gene responses only if prolonged overexpression is non-toxic. Such systems can also lead to the development of compensatory mechanisms within a cell that can mask functional phenotypes (El-Brolosy et al., 2019). Inducible expression systems are a favored alternative to constitutive expression, giving researchers the ability to switch genes on and off or titrate the level of gene expression, with fewer side effects and greater efficiency. One advantage of such conditional expression is the ability to regulate gene expression in a quantitative, temporally-regulated, and reversible manner that more accurately reveals the direct result of a given genetic change. The tetracycline-controlled operator system containing the TRE promoter provides reversible gene expression with a large induction range at low concentrations of a drug that is non-toxic to mammalian cells. The TRE promoter contains a modified Tet response element consisting of seven repeats of a 36-nt sequence that contains the 19-bp Tet operator sequence (TCCCTATCAGTGATAGAGA). This Tet response element is positioned upstream of a minimal CMV promoter sequence, which lacks the enhancer present in the native CMV promoter. Consequently, the TRE promoter is silent in the absence of binding of the tetracycline responsive rtTA3 regulatory protein. Advances in the system have optimized the reverse Tet transactivator (rtTA) component for improved drug sensitivity and activity and, more importantly, to show no activity in the absence of tetracycline or doxycycline (DOX, a tetracycline analog), thus reducing the leakage of the system (Zhou et al., 2006; Yamada et al., 2018). Nevertheless, it should be noted that tetracycline-derived contaminants are often present in cell culture sera; this poses a challenge for Tet-based systems, but this issue can be avoided by using tetracycline-free serum.


Lentiviruses provide an efficient vehicle for facilitating the establishment of inducible gene expression within a cell, particularly because they are able to infect many different dividing and non-dividing cell types (Naldini et al., 1996; Reiser et al., 1996), making lentiviral systems an essential tool for introducing exogenous genes into difficult to transduce targets such as primary cells. Although lentiviral platforms do pose the risk of endogenous gene disruption, the system described here packages all components into a single vector platform so that only a single viral infection per cell is necessary for tet-inducible gene expression (Figure 1A). This eliminates the need for multiple viral infections and limits the possible number of random genome integrations, thereby limiting the chance of off-target effects on cell physiology (Connolly et al., 2002). The constitutively expressed Neo/Venus transduction marker contained in the system allows for easy enrichment of the transduced cell population and can also be easily replaced with another selection marker (e.g., any antibiotic or cell surface marker) to optimize the system for a range of cell types and applications. This not only makes the system highly tractable, but the development of third generation lentiviral vectors, such as pSLIK (Shin et al., 2006), which separate the minimal genetic elements of HIV into three plasmids (pMDL containing gag and pol, pRSV containing the rev protein, and pVSV containing the envelope protein), increases the safety of lentiviruses and decreases the need for highly specialized training (Barde et al., 2010). While this means that the lentivector system described here requires the transfection of four separate plasmids to generate functional lentiviral particles, this design substantially increases its safety for common laboratory use over second generation platforms that expressed the Gag, Pol, Rev, and Tat genes from a single packaging plasmid. Importantly, third generation lentiviral platforms are self-inactivating in nature due to a deletion in the long terminal repeat (LTR) region that results in the loss of the pro-viral enhancer sequence upon integration. This further improves biosafety because replication-competent viruses are not generated within the target cells (Li et al., 2005). Finally, our lentiviral system utilizes a hybrid 5’LTR fused to a CMV promoter that increases gene expression. This is in contrast to second generation systems, which relied on a weak viral 5’ LTR and required the presence of Tat to activate gene expression. Overall, the third generation lentiviral platform described in this protocol ensures robust gene expression from a single infection of cells and increases the laboratory safety of lentiviruses.


Combining inducible expression with such a flexible lentiviral vector platform allows for tetracycline-regulated gene expression from a minimal viral infection of a wide range of cell targets (Shin et al., 2006). We utilized the Tet-on configuration of such a system in our recent paper (Rommereim et al., 2020) to express Nod-like receptors in THP-1 cells and provide the detailed protocol here.

Materials and Reagents

  1. Consumables and reagents

    1. 10× PBS (Gibco, catalog number: 70011044), store at 4°C

    2. 293T culture dishes; 100 × 15 mm TC-treated Petri dishes (Corning Falcon, catalog number: 353003)

    3. Amicon Ultra-15 Centrifugal Filter Units (Millipore, catalog number: UFC903024)

    4. DMEM (Gibco, catalog number: 11995-065), store at 4°C

    5. FBS (Gibco, catalog number: A31605), store at -20°C

    6. G418, Geneticin (Invivogen, catalog number: ant-gn-1), store at 4°C

    7. Glutamine (Gibco, catalog number: 25030149), store at 4°C

    8. HEPES (Gibco, catalog number: 15630-080), store at 4°C

    9. Lipofectamine 2000 (Invitrogen, catalog number: 52887), store at 4°C

    10. Opti-MEM (Gibco, catalog number: 51985-034), store at 4°C

    11. Penicillin G (Sigma, catalog number: P3032), store at 4°C

    12. Polybrene (Sigma, catalog number: H9268)

    13. Poly-L-Lysine Hydrobromide (Sigma, catalog number: P1274), store at 4°C

    14. RPMI (Gibco, catalog number: 11875-101), store at 4°C

    15. Sodium Pyruvate (Gibco, catalog number: 11360-070), store at 4°C

    16. Valmark Ultra-Dish Petri dishes 100 mm × 15 mm (Midwest Scientific, catalog number: 900)


  2. Plasmids

    1. Entry Vector pEN_TmiRc3 (Addgene Plasmid #25748)

      Entry vector for cloning a gene of interest, such that its expression is driven by a TRE promoter.

    2. Lentiviral expression vector pSLIK_Neo (Addgene Plasmid #25735)

      3rd generation lentiviral expression vector that allows for inducible Tet-based gene expression and contains a constitutive neomycin resistance cassette.

    3. pMDL (Addgene Plasmid #12251)

      3rd generation lentiviral packaging plasmid that contains gag and pol.

    4. pRSV (Addgene Plasmid #12253)

      3rd generation lentiviral packaging plasmid that contains rev.

    5. pVSV (Addgene Plasmid #138479)

      Lentiviral packaging plasmid that contains the VSV envelope protein.


  3. Cell lines and media

    1. HEK293T cells (ATCC, catalog number: CRL-3216)

    2. THP-1 cells (ATCC, catalog number: TIB-202)

    3. HEK293T Growth Media (see Recipes)

    4. THP-1 Growth Media (see Recipes)


  4. Primers

    See Table 1


    Table 1. List of primers and probes used for evaluating the expression of endogenous and 3X-FLAG tagged NOD1 and NLRP2 by qPCR

    Gene Name/Product 5’-3’ Sequence Type
    FLAG-NOD1 Exon 3 ATCGATTACAAGGATGACGATGAC Forward Primer
    GGGTGAGACTCTGATGGGATTATT Reverse Primer
    NOD1 Exon 2 GATGGCAAGAGGTGGAGATTG Forward Primer
    TTCCCATAAAAACAGCAACTTGTCT Reverse Primer
    FLAG-NLRP2 Exon 1 ATCGATTACAAGGATGACGATGAC Forward Primer
    CCAGGAGAGCCTGCAGGTT Reverse Primer
    NLRP2 Exon 14 3’-UTR CTCCATGAAGTCATCGATTTTCC Forward Primer
    ACATCTAGCCCAGCAATGAACTC Reverse Primer

Equipment

  1. Centrifuge (Eppendorf, model: 5810R, catalog number: 022625004)

  2. DNA Spectrophotometer (Nanodrop, model: ND-1000, catalog number: THERMO-ND1000)

  3. Flow Cytometer (Becton Dickinson, model: BD FACSCaliburTM)

  4. Gel Imaging System (Bio-Rad, model: ChemiDoc XRS+, catalog number: 1708265)

  5. Protein gel tank (Invitrogen, model: Mini gel tank, catalog number: A25977)

  6. Real-time PCR system (Applied Biosystems, model: Quant Studio 6 Flex, catalog number: 4485691)

  7. Thermal Cycler (Bio-Rad, model: T100, catalog number:1861096)

  8. Transfer apparatus (Bio-Rad, model: Trans-blot SD Semi-Dry Transfer Cell, catalog number: 1703940)

Software

  1. GraphPad Prism Version 9

Procedure

  1. Prepare plates with Poly-L-Lysine

    Note: Poly-L-Lysine enhances cell adhesion, decreasing the probability of HEK293T cells detaching from the plate during transfection.

    1. Dilute 20 mg/ml Poly-L-lysine (PLL) to 20 μg/ml PLL in 1× PBS by combining 50 μl of PLL (20 mg/ml) with 5 ml of 10× PBS and 45 ml of ddH2O.

    2. Add 4 ml of diluted PLL (20 μg/ml) to a 10 cm 293T culture dish.

    3. Incubate for 1 h at 37°C.

    4. Aspirate PLL solution and wash three times with 5 ml of 1× PBS. To store dishes, rinse three times with sterile ddH2O and air dry plates in a sterile tissue culture hood at room temperature.


  2. Passage HEK293T cells

    1. Aspirate media from a T75 flask of HEK293T cells at 90% confluency. One T75 flask should contain enough cells to plate three 10 cm dishes at 4.5 × 106 cells per dish.

    2. Wash flask with 5 ml of 2 mM EDTA PBS.

    3. Add 5 ml of warm Trypsin-EDTA and gently rock the flask to detach cells.

    4. Collect cells in a 15 ml conical tube and add 5 ml of complete media to inhibit trypsin.

    5. Spin at 300 × g for 5 min.

    6. Aspirate media and flick the pellet to loosen cells, resuspending in 10 ml of media and count cells.

    7. Plate 4.5 × 106 cells per PLL-coated 10 cm dish; cells should quickly adhere to the dish.


  3. Transfect Cells

    1. Change media on cells with 10 ml of regular growth media per plate 1 h prior to transfection.

    2. Warm Opti-MEM (OM) at 37°C.

    3. Dilute DNA constructs in OM to a total of 1.5 ml at the amounts listed in Table 2.

      Table 2. Amount of DNA constructs required for lentiviral production

      Plasmid DNA (μg)
      pMDL 7.5
      pRSV 7.5
      pVSV 5
      Expression Plasmid (Figure 1A) 10

    4. Dilute 60 μl of Lipofectamine 2000 (LF2000) with 1.44 ml OM. Let stand at room temperature for 5 min.

    5. Add the 1.5 ml solution of LF2000/OM to the diluted DNA and let stand for 20 min at room temperature.

    6. Carefully add 3 ml of DNA/LF2000/OM to the 10 ml media in the plate, one drop at a time.

    7. Incubate overnight (12-18 h) at 37°C.

    8. Aspirate media and replace with 10 ml of warm culture media per plate.

    9. Incubate for an additional 36 h.


  4. Virus Collection

    1. Transfer media from 10 cm 293T culture plate to a 15 ml conical tube.

    2. Centrifuge supernatant for 5 min at 500 × g at 4°C.

    3. Filter supernatant through a 0.45 μm non-pyrogenic filter.

    4. Store supernatant at 4°C while the Amicon unit is prepared.

    5. Sterilize the Amicon unit by adding 15 ml of 70% ethanol to the filter cup. Let it sit for 10 min.

    6. Centrifuge the Amicon unit for 15 min at 2,590 × g.

    7. Discard ethanol from the collection tube.

    8. Add 15 ml of sterile water to the filter cup. Let it sit for 5 min.

    9. Centrifuge the Amicon unit for 15 min at 2,590 × g. Discard water from the collection tube.

    10. Add the viral supernatant to the Amicon unit.

    11. Centrifuge for 30 min at 2,590 × g at 4°C. The supernatant should completely pass through the filter, with none remaining in the filter cup portion.

      Alternative: If Amicon unit is unavailable, aliquot the supernatant from Step D4 into a 50 ml conical tube and set up an overnight centrifugation. Spin with labels facing out to know where to look for the pellet. Spin with slow acceleration (4) and normal braking (9) at 8,000 × g for 12 to 18 h at 4°C.

    12. Add 1 ml of RPMI to the filter cup. Collect the viscous concentrated virus and store at 4°C.

      Alternative: If concentrating the virus by centrifugation, aspirate the supernatant. Resuspend pellet in 1 ml of RPMI and store at 4°C.


  5. Lentivirus titration in 293T cells

    Notes:

    1. To achieve optimal lentiviral infection of target cells, it is important to use the correct ratio of cells to infectious viral particles. This ratio is also known as the multiplicity of infection (MOI). The number of viable infectious viral particles in the concentrated lentivirus stock is determined from a viral titration experiment. Viral infection can also be scored by flow cytometry to detect the presence of a fluorescent protein co-expressed in the lentiviral construct or staining for a gene encoded by the lentivector. While we describe the use of the pSLIK-Neo viral backbone, which co-expresses a Neomycin resistance gene, a similar pSLIK parental plasmid, which co-expresses the Venus yellow fluorescent protein (Addgene# 25734), is available.

    2. The infectious viral titer is determined in the unconcentrated culture supernatant collected from the packaging cells, the concentrated viral stock, and in the concentrated viral stock following a freeze-thaw cycle. Comparison of the titer of these different preparations aids in the identification of sources of viral loss, which can be an important factor to control.


    1. Plate 293T cells harvested from log phase cultures in 12-well tissue culture plates at a concentration of 1.44 × 105 cells/well and incubate overnight. Visually confirm that the 293T cells are healthy, evenly distributed, and at 40-50% confluence at the time of infection.

    2. Aspirate medium from 293T cells and add 0.9 ml of pre-warmed 293T growth medium containing 8 µg/ml polybrene. Return plates to incubator.

    3. Dilute viral solutions in 293T growth medium containing 8 µg/ml polybrene. A final volume of 100 µl is needed for each dilution, and each sample will be diluted another 10-fold when added to the cells. A set of serial dilutions (usually three) over the range 10-103 is tested for unconcentrated virus, and a range of 102-105 is tested for concentrated virus.

    4. Add 100 µl of each virus dilution to wells of 293T cells (in 0.9 ml of media). To serve as matched uninfected controls, add 100 µl of 293T growth media/polybrene that does not contain virus to several wells. Incubate cells for 24 h.

    5. After 24 h, replace the media with fresh 293T growth medium and return plates to the incubator.

    6. Harvest cells for flow cytometric analysis 48-72 h after infection. Pool the media supernatant, washes, and dislodged cells from each sample well into FACS tubes to ensure that all cells are included.

    7. Pellet cells by centrifugation (300 × g, 5 min, 4°C).

    8. Determine the percentage of Venus-positive cells by flow cytometric analysis by comparison of infected samples with uninfected 293T cells. Identify samples in which the infection rate is 3-10% and calculate the number of cells infected on the day of infection. This is approximately equivalent to the number of infectious virus particles added per well, assuming a 1:1 infection ratio. The number of active viruses detected is used with the volume and dilution of virus stock to calculate the virus concentration in the stock solution. This provides the lentiviral titer with respect to 293T cell infection.

      Lentiviral titer = number of infected cells × volume of virus stock × dilution of virus stock

      Notes:

      1. The titer for the user’s target cells of interest can be calculated by side-by-side infection of such target cells, with 293T cells using identical cell numbers and viral quantity. Many cell types may infect less efficiently than 293T cells, but we have found the relative infection ratio between cells to be quite consistent. Thus, if the user’s target cells typically show 50% of the 293T infection level, this ratio can be assumed for future viral preparations.

      2. Similarly, we have found side-by-side comparison of the pSLIK-Venus and pSLIK-Neo parental plasmids to yield very similar viral titers. Thus, titration of a pSLIK-Venus control alongside pSLIK-Neo based viruses should yield comparable titers.

      Our average viral titer from this protocol is 1.175 × 107 viral particles from 1 plate of 293T cells. Although each lab should carry out several viral titrations to determine the level and consistency of their titers (Kutner et al., 2009), we use this average value for THP-1 cell infections and only run titrations if we find our infection rates drop unexpectedly.


  6. THP-1 Cell Transduction

    1. Calculate viral particles per milliliter by dividing 1.175 × 107 by the total volume of concentrated virus recovered per plate.

    2. Calculate the volume of virus needed to infect cells at 10 MOI by dividing 5.0 × 106 by the concentration of viral particles per ml. We typically infect 0.5 × 106 THP-1 cells with an approximate MOI of 10 (293T transduction units).

      Volume of virus (ml) = (number of cells per well × MOI)/viral particles per ml, i.e.

      (0.5 × 106 × 10)/viral particles per ml.

      This equates to an MOI of <1 for THP-1 cells, usually around 30% transduction efficiency [where transduction efficiency (%) = number of transduced cells/total number of cells]. Transduction efficiency can be measured by FACS to count the number of THP-1 cells that are positive for expression of the lentivirus-encoded transgene or a constitutively expressed Venus transduction marker as a fraction of the total number of cells. We aim for this relatively low infectivity to ensure that most cells do not have multiple viral integrations.

    3. Plate 0.5 × 106 cells per well into two wells of a 24-well dish.

    4. Infect cells 0.5-3 h after seeding.

    5. Infect one well by diluting virus up to 500 μl with serum-free growth medium supplemented with 4 μg/ml polybrene and 100 U/ml penicillin G.

    6. Aspirate media and replace it with diluted virus.

    7. After 4 h, add 1.5 ml regular growth media with penicillin.

    8. The next day, split cells and add 1 × 106 cells into Valmark dishes.

    9. One to two days after splitting, select for cells by growing in complete media supplemented with G418 (1 mg/ml).


  7. Gene Expression Validation in THP-1 Cells

    1. Plate cells in a 6-well plate and incubate at 37°C for 6 h.

    2. Add DOX to the cells (1 μg/ml) and incubate for 6-18 h.

    3. Harvest cells and isolate RNA or protein.

    4. Validate viral integration by qPCR (Figure 1B) and DOX-induced overexpression by qPCR, immunoblot, or flow cytometry (Figure 1C-1E).



      Figure 1. Lentivirus-mediated conditional expression of NOD1 and NLRP2. (A) Lentiviral system for DOX-inducible expression of NLRs. Expression of 3X-FLAG-tagged NLR coding sequence was transcribed under the control of an inducible tetracycline promoter, called a tetracycline response element (TRE). To engineer the lentiviral expression vectors pSLIK_Neo_NOD1 or pSLIK_Neo_NLRP2, N-terminal 3X-FLAG-tagged NOD1 or NLRP2 were first cloned between the Spe1 and Mfe1 restriction sites of the entry vector pEN_TmiRc3, so that their gene expression was driven by a TRE promoter. Entry vectors were then recombined with the lentiviral expression vector, pSLIK_Neo, via the Gateway LR cloning reaction. This recombination reaction shuttles the TRE-driven NOD1 and NLRP2 gene cassettes into the lentiviral backbone, resulting in the generation of pSLIK_Neo_NOD1 and pSLIK_Neo_NLRP2 lentiviral expression vectors. Lentiviral plasmids were co-transfected with pMDL, pRSV, and pVSV into HEK293T cells to generate lentiviruses used to infect THP-1 cells. (B) qPCR showing construct:chromosome ratio of the conditionally expressed NLRs. THP-1 cells were infected with lentivirus at low infectivity. qPCR probes specific for the endogenous or the FLAG-tagged NLR (Table 1) were used to determine the ratio of the NLR transgene (construct) to the endogenous gene (chromosome), as an estimate of the number of integrations per genome. A construct:chromosome ratio of 0.5 indicates one integration per cell, and a ratio of 1 indicates two integrations per cell. (C) qPCR showing overexpression of NLRs upon DOX addition. THP-1 cells harboring DOX-inducible NOD1 or NLRP2 were left untreated or were treated with DOX for 6 h. RNA was harvested, and NLR expression was determined by qPCR. NLR expression in the absence of DOX was set at 1. DOX-induced NLR overexpression was measured relative to that in the absence of DOX. (D and E) Immunoblot (D) and flow cytometry (E) showing protein expression of FLAG-tagged NLRs in the indicated THP-1 lines with or without addition of DOX for 6 h. ‘Vector’ refers to cells transduced with empty vector and ‘control’ refers to THP-1 cells with no lentiviral transduction. Data are presented as means ± SEM. Statistical analyses were performed using a Student’s unpaired t-test (two-tailed) in GraphPad Prism. P values <0.05 were considered significant. ****P < 0.0001.

Recipes

  1. HEK293T Growth Media

    DMEM supplemented with 10% FBS, 10 mM HEPES, and 2 mM glutamine.

  2. THP-1 Growth Media

    RPMI supplemented with 10% FBS, 10 mM HEPES, and 2 mM glutamine

Acknowledgments

This work was funded by the ISB, U.S. NIH grant R21-AI138258 to N.S. and the Intramural Research Program of NIAID, NIH (R.N.G and I.D.C.F). A.S.A. was supported by an American Association of Immunologists (AAI) Careers in Immunology Fellowship. We thank S. Porcella and the Genomics Unit at Rocky Mountain Laboratories for primer design and qPCR support. This protocol was adapted with minor modifications from Shin et al. (2006) and Rommereim et al. (2020).

Competing interests

Leah Rommereim is an employee and stockholder of SEngine Precision Medicine. None of the other authors have any competing financial interests.

References

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简介

[摘要]识别系统内特定基因作用的能力取决于对该基因的表达。在这个协议中,我们描述了一种稳定的条件表达方法使用慢病毒表达系统的 THP-1 细胞中的 Nod 样受体 (NLR)。该系统结合四环素诱导基因在单个慢载体中表达的所有必要成分选择标记的组成型共表达,这是控制基因的有效手段使用细胞的单一病毒感染来表达。这是在第三代慢病毒表达中完成的提高慢病毒安全性并允许比以前更高的基因表达的平台慢病毒平台。慢病毒表达质粒首先被设计为包含感兴趣的基因在简单的网关克隆步骤中由 TRE(四环素反应元件)启动子驱动,然后与包装和包膜质粒共转染到 HEK293T 细胞中以产生病毒。该病毒用于以低 MOI 感染感兴趣的细胞类型,以便大多数转导细胞包含单个病毒整合。受感染的细胞在选择下生长,病毒整合是通过 qPCR 验证。稳定转导细胞中的基因表达由强力霉素和通过 qPCR、免疫印迹和流式细胞术验证。这种灵活的慢病毒表达平台可能是用于在一系列细胞中稳定和稳健地诱导感兴趣的基因,以用于多种应用。

图文摘要:THP-1 细胞慢病毒转导示意图。

关键词: NLR,NOD1,慢病毒,稳定表达,TRE,四环素

[背景]在分子生物学中研究功能反应的一项关键技术是来调节基因的表达。组成型基因表达系统允许研究人员只有在长期过表达无毒的情况下才能探索基因反应。这样的系统也可以导致细胞内代偿机制的发展,可以掩盖功能表型(El-Brolosy等人。, 2019)。诱导型表达系统是组成型表达的首选替代方案,使研究人员能够打开和关闭基因或滴定基因表达水平,更少的副作用和更高的效率。这种条件表达式的一个优点是能够以定量、时间调节和可逆的方式调节基因表达准确地揭示给定遗传变化的直接结果。四环素控制算子包含 TRE 启动子的系统提供具有大诱导范围的可逆基因表达低浓度的对哺乳动物细胞无毒的药物。TRE 启动子包含一个修改的 Tet 响应元件由包含 19-bp 的 36-nt 序列的七个重复组成Tet 运算符序列 (TCCCTATCAGTGATAGAGA)。这个 Tet 响应元素被定位最小 CMV 启动子序列的上游,该序列缺少天然 CMV 中存在的增强子发起人。因此,TRE 启动子在不与四环素结合的情况下是沉默的反应性 rtTA3 调节蛋白。系统的进步优化了反向Tet反式激活剂 (rtTA) 成分可提高药物敏感性和活性,更重要的是,显示在没有四环素或强力霉素(DOX,四环素类似物)的情况下没有活性,从而减少系统泄漏(Zhou et al. , 2006; Yamada et al. , 2018)。尽管如此,应该指出的是四环素衍生的污染物通常存在于细胞培养血清中;这对 Tet 提出了挑战-基于系统,但这个问题可以通过使用不含四环素的血清来避免。慢病毒为促进诱导基因表达的建立提供了有效的载体在一个细胞内,特别是因为它们能够感染许多不同的分裂和非分裂细胞类型(Naldini等人,1996 年;Reiser等人,1996 年),使慢病毒系统成为引入外源基因进入难以转导的目标,如原代细胞。虽然慢病毒平台确实存在内源性基因破坏的风险,这里描述的系统包装了所有组件进入单个载体平台,以便每个细胞只需要一次病毒感染即可诱导 tet基因表达(图 1A)。这消除了对多种病毒感染的需要并限制了可能的随机基因组整合的数量,从而限制对细胞的脱靶效应的机会生理学(Connolly等人,2002 年)。组成型表达的 Neo/Venus 转导标记包含在系统中可以轻松富集转导的细胞群,也可以很容易用另一种选择标记(例如,任何抗生素或细胞表面标记)替换以优化适用于各种细胞类型和应用的系统。这不仅使系统高度易于处理,而且第三代慢病毒载体的开发,如 pSLIK (Shin et al ., 2006),它分离了将 HIV 的最小遗传元件分成三个质粒(pMDL 含有 gag 和 pol,pRSV 含有rev 蛋白和含有包膜蛋白的 pVSV),增加了慢病毒的安全性和减少了对高度专业化培训的需求(Barde等,2010)。虽然这意味着此处描述的慢载体系统需要转染四个单独的质粒以产生功能性慢病毒颗粒,这种设计大大提高了实验室常用的安全性在第二代平台上表达 Gag、Pol、Rev 和 Tat 基因包装质粒。重要的是,第三代慢病毒平台在本质上是自我灭活的,因为长末端重复 (LTR) 区域的缺失导致原病毒增强子的丢失整合后的顺序。这进一步提高了生物安全性,因为具有复制能力的病毒是不在靶细胞内生成(Li et al ., 2005)。最后,我们的慢病毒系统利用混合 5'LTR与增加基因表达的 CMV 启动子融合。这与二代相反系统,它依赖于弱病毒 5' LTR 并需要 Tat 的存在来激活基因表达。总体而言,本协议中描述的第三代慢病毒平台确保稳健单次细胞感染的基因表达,提高了慢病毒的实验室安全性。将诱导型表达与这种灵活的慢病毒载体平台相结合,可以实现四环素-来自广泛细胞靶标的最小病毒感染调节基因表达(Shin等,2006)。我们在最近的论文 (Rommereim et al. , 2020) 中利用了此类系统的 Tet-on 配置来在 THP-1 细胞中表达 Nod 样受体,并在此处提供详细的实验方案。

关键字:NLR, NOD1, 慢病毒 , 稳定表达 , TRE, 四环素

材料和试剂
A. 耗材和试剂
1. 10×PBS(Gibco,目录号:70011044),4°C保存
2、293T培养皿;100 × 15mm TC 处理的培养皿(Corning Falcon,目录号:
353003)
3. Amicon Ultra-15离心过滤装置(Millipore,目录号:UFC903024)
4. DMEM(Gibco,目录号:11995-065),4°C 保存
5. FBS(Gibco,目录号:A31605),储存在-20°C
6. G418,遗传霉素(Invivogen,目录号:ant-gn-1),4°C 保存
7. 谷氨酰胺(Gibco,目录号:25030149),在 4°C 下储存
8. HEPES(Gibco,目录号:15630-080),在 4°C 下储存
9. Lipofectamine 2000(Invitrogen,目录号:52887),在 4°C 下储存
10. Opti-MEM(Gibco,目录号:51985-034),在 4°C 下储存
11. 青霉素 G(Sigma,目录号:P3032),储存在 4°C
12.聚凝胺(Sigma,目录号:H9268)
13.聚-L-赖氨酸氢溴酸盐(Sigma,目录号:P1274),储存在4°C
14. RPMI(Gibco,目录号:11875-101),在 4°C 下储存
15. 丙酮酸钠(Gibco,目录号:11360-070),储存在 4°C
16. Valmark Ultra-Dish Petri 培养皿 100mm x 15mm(Midwest Scientific,目录号:900)
B. 质粒
1. 入口载体 pEN_TmiRc3 (Addgene Plasmid #25748)
用于克隆感兴趣基因的入口载体,使其表达由 TRE 启动子驱动。
2. 慢病毒表达载体 pSLIK_Neo (Addgene Plasmid #25735)
3次代慢病毒表达载体,其允许可诱导的基于的Tet基因表达
并包含组成型新霉素抗性盒。
3. pMDL (Addgene Plasmid #12251)
3次代慢病毒包装质粒包含gag和pol。
4. pRSV (Addgene Plasmid #12253)
3次代慢病毒包装质粒含有转。
5. pVSV (Addgene Plasmid #138479)
包含 VSV 包膜蛋白的慢病毒包装质粒。
C. 细胞系和培养基
1. HEK293T 细胞(ATCC,目录号:CRL-3216)
2. THP-1 细胞(ATCC,目录号:TIB-202)
3. HEK293T 生长培养基(见配方)
4. THP-1 生长培养基(见食谱)
D. 引物
见表 1


设备
1.离心机(Eppendorf,型号:5810R,目录号:022625004)
2. DNA分光光度计(Nanodrop,型号:ND-1000,目录号:THERMO-ND1000)
3.流式细胞仪(Becton Dickinson,型号:BD FACSCalibur TM)
4.凝胶成像系统(Bio-Rad,型号:ChemiDoc XRS+,目录号:1708265)
5.蛋白质凝胶罐(Invitrogen,型号:Mini gel tank,目录号:A25977)
6. 实时 PCR 系统(Applied Biosystems,型号:Quant Studio 6 Flex,目录号:
4485691)
7.热循环仪(Bio-Rad,型号:T100,目录号:1861096)
8. 转移装置(Bio-Rad,型号:Trans-blot SD Semi-Dry Transfer Cell,目录号:
1703940)
软件
1. GraphPad Prism 版本 9
程序
A. 用聚-L-赖氨酸准备板
注:Poly-L-Lysine 增强细胞粘附,降低 HEK293T 细胞脱离的可能性
在转染过程中从板中取出。
1. 将 50 μl PLL (20
mg/ml) 与 5 ml 10× PBS 和 45 ml ddH 2 O。
2. 将 4 ml 稀释的 PLL (20 μg/ml) 添加到 10 cm 293T 培养皿中。
3. 37°C 孵育 1 小时。
4. 吸出 PLL 溶液并用 5 ml 1× PBS 洗涤 3 次。存放餐具,冲洗三
在室温下,在无菌组织培养罩中使用无菌 ddH 2 O 和空气干燥板。
B. 传代 HEK293T 细胞
1. 从 HEK293T 细胞的 T75 烧瓶中以 90% 汇合度吸出培养基。一个 T75 烧瓶应该
包含足够的细胞以每盘4.5 × 10 6细胞培养三个 10 cm培养皿。
2. 用 5 ml 2 mM EDTA PBS 清洗烧瓶。
3. 加入 5 ml 温热的胰蛋白酶-EDTA 并轻轻摇动烧瓶以分离细胞。
4. 在 15 ml 锥形管中收集细胞并加入 5 ml 完全培养基以抑制胰蛋白酶。
5. 以 300 × g旋转5 分钟。
6. 吸出培养基并轻弹沉淀以松开细胞,重悬于 10 ml 培养基中并计数
细胞。
7.每个 PLL-coated 10 cm 培养皿中平板 4.5 × 10 6 个细胞;细胞应迅速粘附在培养皿上。
C. 转染细胞
1. 在转染前 1 小时用 10 ml 常规生长培养基每板更换细胞培养基。
2. 在 37°C 下加热 Opti-MEM (OM)。
3. 将 OM 中的 DNA 构建体按表 2 中所列的量稀释至总计 1.5 ml。
与1.44毫升OM脂质体2000(LF2000)的4稀释60微升。让我们在室温下静置
5 分钟。
5. 将 1.5 ml LF2000/OM 溶液加入稀释的 DNA 中,室温静置 20 分钟
温度。
6.小心添加3毫升DNA / LF2000 / OM的到10 ml培养基在板上,在每次一滴。
7. 在 37°C 下孵育过夜(12-18 小时)。
8. 吸出培养基并更换为每板 10 毫升温热培养基。
9. 再孵育 36 小时。
D. 病毒收集
1. 将培养基从 10 cm 293T 培养板转移到 15 ml 锥形管中。
2. 将上清液在 4°C 下以 500 × g离心 5 分钟。
3. 通过 0.45 μm 无热原过滤器过滤上清液。
4. 准备好 Amicon 装置时,将上清液储存在 4°C。
5. 通过向滤杯中加入 15 ml 70% 乙醇对 Amicon 装置进行消毒。让它静置10分钟。
6. 将 Amicon 装置以 2,590 × g离心 15 分钟。
7. 丢弃收集管中的乙醇。
8. 向滤杯中加入 15 毫升无菌水。让它静置 5 分钟。
9.离心的Amicon单元在2590 15分钟×克。从收集管弃水。
10.病毒上清液添加到所述的Amicon单元。
11.在 4°C 下以 2,590 × g离心 30 分钟。上清液应完全通过
过滤器,滤杯部分没有残留。
替代方法:如果的Amicon单元不可用,从等分步骤D4上清液至50ml
锥形管并设置一个过夜离心。自旋与朝外标签,知道在哪里
寻找颗粒。以 8,000 × g缓慢加速 (4) 和正常制动 (9) 旋转12
4°C 至 18 小时。
12. 向滤杯中加入 1 ml RPMI。收集粘稠的浓缩病毒并在 4°C 下储存。
替代方法:如果通过离心浓缩病毒,吸出上清液。重悬
在 1 ml RPMI 中沉淀并在 4°C 下储存。
E. 293T 细胞中的慢病毒滴定
笔记:
一种。为了实现靶细胞的最佳慢病毒感染,重要的是使用正确的比例细胞转化为感染性病毒颗粒。该比率也称为感染复数 (MOI)。确定浓缩慢病毒原液中活的传染性病毒颗粒的数量来自病毒滴定实验。病毒感染也可以通过流式细胞术进行评分以检测慢病毒构建体中共表达的荧光蛋白的存在或染色基因编码由慢病毒载体。虽然我们描述了 pSLIK-Neo 病毒骨架的使用,它共表达了一个新霉素抗性基因,一个类似的pSLIK 亲本质粒,它共表达金星黄色荧光蛋白 (Addgene# 25734),可用。湾 感染性病毒滴度是在未浓缩的培养上清液中测定的包装细胞、浓缩病毒原液和浓缩病毒原液冻融循环。比较这些不同制剂的效价有助于确定病毒损失的来源,这可能是一个重要的控制因素。
1.在12孔组织培养板从对数期培养物中收获板293T细胞
1.44 × 10 5 个细胞/孔的浓度并孵育过夜。目视确认 293T
细胞健康,分布均匀,感染时融合度为 40-50%。
2. 从 293T 细胞中吸出培养基,加入 0.9 ml 预热的 293T 生长培养基,其中含有
8微克/ ml聚凝胺。返回板到培养箱中。
3.稀释在含8微克/ ml聚凝胺293T生长培养基病毒解决方案。最终体积
每次稀释需要 100 µl,每个样品加入时会再稀释 10 倍
到细胞。对 10-10 3范围内的一组连续稀释(通常为三个)进行测试
未浓缩的病毒,10 2 -10 5的范围用于检测浓缩病毒。
4.将100μl各病毒稀释液添加到293T细胞的孔中(在0.9毫升培养基)。作为匹配
未感染的对照,添加 100 µl 不含病毒的 293T 生长培养基/聚凝胺
几口井。孵育细胞 24 小时。
5. 24 小时后,用新鲜的 293T 生长培养基更换培养基并将培养板放回培养箱。
6. 感染后 48-72 小时收获细胞用于流式细胞术分析。汇集培养基上清液,
洗涤,并将每个样品孔中的细胞移入 FACS 管中,以确保所有细胞都
包括。
7. 离心沉淀细胞(300 × g,5 分钟,4°C)。
8. 通过流式细胞术分析确定金星阳性细胞的百分比
受感染的样品未感染293T细胞。确定样品中的感染率是3-
10% 并计算感染当天感染的细胞数。这大约是
相当于每孔添加的传染性病毒颗粒的数量,假设感染率为 1:1
比率。检测到的活性病毒数量与病毒原液的体积和稀释度一起使用
计算原液中的病毒浓度。这提供了慢病毒滴度
关于 293T 细胞感染。
慢病毒滴度 = 感染细胞数 × 病毒原液体积 × 病毒原液稀释度
笔记:
一种。用户感兴趣的目标细胞的滴度可以通过并排感染来计算此类靶细胞,293T 细胞使用相同的细胞数和病毒量。许多细胞类型可能比 293T 细胞感染效率低,但我们发现相对感染细胞之间的比例相当一致。因此,如果用户的目标单元格通常显示 50%的293T感染水平的,可以假设以供将来病毒制剂这个比例。湾 同样,我们发现了 pSLIK-Venus 和 pSLIK-Neo 的并排比较亲本质粒产生非常相似的病毒滴度。因此pSLIK-Venus 对照的滴定与基于 pSLIK-Neo 的病毒一起应产生可比的滴度。
我们来自该协议的平均病毒滴度是来自 1 块 293T 细胞的1.175 × 10 7病毒颗粒。虽然每个实验室都应该进行几次病毒滴定以确定水平和一致性的效价 (Kutner et al ., 2009),我们将这个平均值用于 THP-1 细胞感染,并且仅如果我们发现感染率意外下降,请进行滴定。
F. THP-1 细胞转导
1. 用 1.175 × 10 7除以浓缩的总体积计算每毫升病毒粒子数
每板回收病毒。
2.计算在10 MOI需要感染细胞的病毒通过将5.0×10容积6由
每毫升病毒颗粒的浓度。我们通常用 0.5 × 10 6 THP-1 细胞感染
大约 MOI 为 10(293T 转导单位)。
病毒体积 (ml) = (每孔细胞数 × MOI)/每毫升病毒颗粒,即。
(0.5 × 10 6 × 10)/每毫升病毒颗粒。
这相当于 THP-1 细胞的 MOI <1,通常约为 30% 的转导效率 [其中转导效率 (%) = 转导细胞数/细胞总数]。转导效率可以通过FACS来测定计数是阳性的THP-1细胞的数目慢病毒编码的转基因或组成型表达的金星转导的表达标记作为细胞总数的一部分。我们的目标是实现这种相对较低的传染性,以确保大多数细胞没有多重病毒整合。
3. 将每孔0.5 × 10 6细胞接种到 24 孔培养皿的两个孔中。
4. 接种后 0.5-3 小时感染细胞。
5. 用无血清培养基稀释病毒至 500 μl 感染一个孔
4微克/ ml聚凝胺和100U / ml青霉素G.
6. 吸出培养基并用稀释的病毒代替。
7. 4 小时后,加入 1.5 ml 含有青霉素的常规生长培养基。
8. 第二天,分裂细胞并将1 × 10 6 个细胞加入Valmark 培养皿中。
9. 分裂后一到两天,通过在补充的完全培养基中生长来选择细胞
与 G418 (1 毫克/毫升)。
在THP-1细胞G.基因表达验证
1. 将细胞置于 6 孔板中,37°C 孵育 6 小时。
2. 向细胞中加入 DOX (1 μg/ml) 并孵育 6-18 小时。
3. 收获细胞并分离 RNA 或蛋白质。
4.验证病毒整合通过qPCR(图1B)和DOX诱导的过量表达通过qPCR,
免疫印迹,或流式细胞术(图1C-1E)。


图 1. NOD1 和 NLRP2 的慢病毒介导的条件表达。慢病毒NLR 的 DOX 诱导表达系统。3X-FLAG-tagged NLR编码的表达序列在诱导型四环素启动子的控制下转录,称为 a四环素响应元件(TRE)。为了设计慢病毒表达载体pSLIK_Neo_NOD1 或pSLIK_Neo_NLRP2,N 端 3X-FLAG 标记的 NOD1 或 NLRP2首先克隆在进入载体 pEN_TmiRc3 的 Spe1 和 Mfe1 限制性位点之间,所以它们的基因表达是由TRE启动子驱动。然后重组入口载体使用慢病毒表达载体 pSLIK_Neo,通过 Gateway LR 克隆反应。这个重组反应将 TRE 驱动的 NOD1 和 NLRP2 基因盒穿梭到慢病毒骨架,导致产生 pSLIK_Neo_NOD1 和 pSLIK_Neo_NLRP2慢病毒表达载体。慢病毒质粒与 pMDL、pRSV 和pVSV 进入 HEK293T 细胞以生成用于感染 THP-1 细胞的慢病毒。( B ) qPCR 显示构建体:条件表达的 NLR 的染色体比率。THP-1细胞被感染慢病毒感染性低。特定于内源性或 FLAG 标记的 qPCR 探针NLR(表 1)用于确定 NLR 转基因(构建体)与内源基因(染色体),作为每个基因组整合数的估计。一种构建体:染色体比率为 0.5 表示每个细胞有一个整合,比率为 1 表示每个单元有两个集成。(C)qPCR 显示添加DOX后NLR 的过度表达。THP-含有 DOX 诱导型NOD1或NLRP2 的1 个细胞未经处理或用 DOX 处理6 小时。收获 RNA,并通过 qPCR 确定 NLR 表达。NLR表达在没有 DOX 的情况下设置为 1。 DOX 诱导的 NLR 过表达是相对测量的在没有 DOX 的情况下。( D和E ) 免疫印迹 (D) 和流式细胞术 (E) 显示蛋白质添加或不添加 DOX 的指定 THP-1 系中 FLAG 标记的 NLR 的表达6 小时。“载体”是指用空载体转导的细胞,“对照”是指 THP-1 细胞没有慢病毒转导。数据表示为平均值±SEM。统计分析使用GraphPad Prism 中的学生未配对t检验(双尾)进行。P值 <0.05被认为是重要的。**** P < 0.0001。


食谱
1. HEK293T 生长培养基DMEM 补充有 10% FBS、10 mM HEPES 和 2 mM 谷氨酰胺。
2. THP-1 生长培养基RPMI 补充有 10% FBS、10 mM HEPES 和 2 mM 谷氨酰胺


致谢
这项工作由 ISB、美国 NIH 向 NS 和校内研究提供 R21-AI138258 资助NIAID、NIH(RNG 和 IDCF)的计划。ASA 得到了美国协会的支持免疫学家 (AAI) 免疫学奖学金职业。我们感谢 S. Porcella 和 Genomics落基山实验室的单位,用于引物设计和 qPCR 支持。该协议是改编自 Shin等人的微小修改。(2006) 和 Rommereim等人。(2020)。


利益争夺
Leah Rommereim 是 SEngine Precision Medicine 的员工和股东。其他都没有作者有任何相互竞争的经济利益。


参考
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Rommereim, L., Akhade, A. S., Germain, R. N., Fraser, I. D. C. and Subramanian, N. (2021). Lentivirus-mediated Conditional Gene Expression . Bio-protocol 11(21): e4205. DOI: 10.21769/BioProtoc.4205.
  2. Rommereim, L. M., Akhade, A. S., Dutta, B., Hutcheon, C., Lounsbury, N. W., Rostomily, C. C., Savan, R., Fraser, I. D. C., Germain, R. N. and Subramanian, N. (2020). A small sustained increase in NOD1 abundance promotes ligand-independent inflammatory and oncogene transcriptional responses. Sci Signal 13(661): eaba3244.
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