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Generation of Luciferase-expressing Tumor Cell Lines
表达萤光素酶的肿瘤细胞系的生成   

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The Journal of Clinical Investigation
Jan 2016

Abstract

Murine tumor models have been critical to advances in our knowledge of tumor physiology and for the development of effective tumor therapies. Essential to these studies is the ability to both track tumor development and quantify tumor burden in vivo. For this purpose, the introduction of genes that confer tumors with bioluminescent properties has been a critical advance for oncologic studies in rodents. Methods of introducing bioluminescent genes, such as firefly luciferase, by viral transduction has allowed for the production of tumor cell lines that can be followed in vivo longitudinally over long periods of time. Here we describe methods for the production of stable luciferase expressing tumor cell lines by lentiviral transduction.

Keywords: Lentivirus (慢病毒), Tumor (肿瘤), Lymphoma (淋巴瘤), Leukemia (白血病), Luciferase (荧光素酶), GFP (GFP), Mouse (小鼠), Methods (方法)

Background

Paramount to tracking cells in vivo is the ability to detect them externally by minimally invasive methods. Enzymatic bioluminescence using luciferase derived from the firefly (Photinus pyralis) is a widely used method for image-based cell tracking in vivo. Bioluminescence has been used for a variety of in vivo application including the noninvasive imaging of reporter gene expression (Herschman, 2004), studying circadian rhythms (Southern and Millar, 2005), imaging cerebral strokes (Vandeputte et al., 2014), and for tracking genetically engineered T cells (Costa et al., 2001; Cheadle et al., 2010). Perhaps the field where bioluminescent cell lines have been most applicable is oncology where they have been instrumental for the monitoring tumor growth (Jenkins et al., 2005; Brennan et al., 2016; Byrne et al., 2016) and tumor metastasis (Rosol et al., 2003; Simmons et al., 2015) in mouse models. While some subcutaneously implanted tumors can be detected by palpation and measured with calipers, these methods are not effective for monitoring metastases or tracking tumors that disseminate widely, such as hematological malignancies that commonly grow in the bone marrow, lymph nodes and spleen.

Firefly luciferase oxides luciferin in the presence of molecular oxygen, magnesium and adenosine triphosphate to produce yellow-green light at 560 nm (Wilson and Hastings, 1998; Fraga, 2008). Benefits of luciferase bioluminescence in cell tracking include penetration of tissue for non-invasive monitoring and the re-usability of the enzymatic marker. Another advantage of luciferases is that most cells are not luminescent such that high signal-to-noise ratios can be achieved. A limitation bioluminescence is photon attenuation caused by intervening tissues, such as skin, bone, or hair.

Firefly luciferase is a single polypeptide specified by the luc gene that can be readily cloned into vectors used in gene delivery. Transient expression by plasmid transfection or non-integrating virus transduction limits the time over which cell tracking can be performed. This is especially problematic for oncology studies that may last several months. The ability of retroviruses to integrate into the genome is a key attribute that favors their use in producing stable cell lines. However, some oncoretroviruses, such as the Moloney murine leukemia virus can be limited by transgene silencing over time (Jähner et al., 1982). Further, retroviral vectors require cell division for genomic integration and can be inefficient at transducing highly differentiated cells such as neurons, dendritic cells, or resting lymphocytes.

For the purpose of making luciferase expressing cell lines, lentiviral retroviral vectors derived from human immunodeficiency virus-1 (HIV-1) are highly effective. An advantage of lentiviral vectors over other retroviral vectors, is their ability to integrate into the genome of non-dividing cells. This property makes them suitable gene delivery vehicles for targeting highly differentiated cells, such as neurons, dendritic cells and lymphocytes (Naldini et al., 1996). Lentiviruses also deliver very stable genomic integration and long-term transgene expression, to the extent that they have been used to make transgenic mice following embryo transduction (Lois et al., 2002).

In order to track hematologic tumor cells in an in vivo murine leukemia model, we made the FULGW lentiviral vector that co-expresses firefly luciferase (Luc) and enhanced green fluorescent protein (EGFP) for the purpose of B-cell lymphoma (A20) cell line transduction and stable clone production. The FULGW vector is based on a self-inactivating vector previously described by Miyoshi et al. (1998) that had been engineered to express the GFP reporter gene behind the human ubiquitin-C promoter by Lois et al. (2002), making the FUGW vector. To make FULGW, we replaced the EGFP sequence of FUGW with a Luc-IRES-EGFP sequence from rKat.Luc2.IRES.EGFP, previously developed by Cheadle et al. (2010).

FULGW contains unique elements that enhance gene integration and expression (Figure 1A). It encodes the human immunodeficiency virus-1 (HIV-1) flap element, giving it karyotropic properties that permit efficient genomic integration in non-replicating cells (Zennou et al., 2000). It contains the wood-chuck hepatitis virus posttranscriptional regulatory element (WPRE) that increases gene expression by transcript stabilization (Zufferey et al., 1999). In addition, it includes a 3’ self-inactivating long terminal repeat (3’ si-LTR) that contributes to maintaining it as a replication deficient virus. The 3’ si-LTR was developed by the deletion of a 133 bp region in the U3 region (ΔU3) of the 3’ LTR that renders the 5’ LTR of the integrated provirus transcriptionally inactive (Miyoshi et al., 1998).

Virus production is performed by the co-transfection of HEK-293T cells with the lentiviral plasmid (FULGW) and the two packaging plasmids, pCMV-ΔR8.91 and pCMV-VSVG (Figure 1B). HEK-293T cells are a human embryonic kidney cell line that stably expresses the CMV large T antigen, which greatly increases gene expression by the CMV promoter, generating robust virus production. pCMVΔR8.91 is an HIV-1 Gag and Polymerase (Pol) expression plasmid that was modified from the dR8.9 vector by deletion of four accessory HIV-1 gents, Vif, Vpr, Vpu, and Nef (Zufferey et al., 1997). pCMV-VSVG expresses the pantropic envelop (Env) protein derived from the vesicular stomatitis virus glycoprotein (VSVG) (Stewart et al., 2003). [*Note: Both FULGW and pCMV-ΔR8.91 are large plasmids and best grown in chemically competent recA1-deficient E. coli with high transformation efficiency such as One Shot TOP10 E. coli (Invitrogen) grown at 30 °C for 24-28 h]. Using the FULGW lentiviral vector packaged with these helper plasmids, we have produced multiple types of tumor cell lines on various genetic backgrounds that stably express luciferase and GFP for use in oncologic studies (Table 1).


Figure 1. Production of FULGW lentivirus and transduction of tumor cell lines. A. Diagram of key regions of the FULGW vector including the Luc-IRES-EGFP transgene. Transgene expression is driven by the human ubiquitin-C promoter. CMV (cytomegalovirus promoter), U5 (LTR unique 5’ region), R (LTR repeat region), HIV-1 flap (human immunodeficiency virus-1 flap element), Luc (firefly luciferase), IRES (intra-ribosomal element sequence), EGFP (enhanced green fluorescent protein), WPRE (wood-chuck hepatitis virus posttranscriptional regulatory element), si-LTR (self-inactivating LTR). B. FULGW is packaged and pseudotyped by lipophilic co-transfecting with pCMV-ΔR8.91 and pCMV-VSVG. Virus rich culture supernatant (SN) is collected at 48 and 72 h and virus is concentrated by ultracentrifugation. The concentrated virus is used to transduce tumor cell lines by spin-transduction in the presence of polybrene. Illustrated schematics make use of Motifolio templates (www.motifolio.com/).

Table 1. Luciferase-GFP expressing tumor cell lines produced by FULGW transduction

Materials and Reagents

  1. Pipette tips (USA Scientific, catalog numbers: 1110-3000 , 1110-1000 , 1111-2021 )
  2. T75 flask (Corning, Falcon®, catalog number: 353136 )
  3. 100 mm TC-treated Tissue Culture Dish (Corning, Falcon®, catalog number: 353003 )
  4. Sterile syringe 0.45 μm filter (VWR, catalog number: 28145-505 )
  5. Beckman ultra-clear 25 x 89 mm tubes (Beckman Coulter, catalog number: 344060 )
  6. Centricon Plus-70 unit (Merck, catalog number: UFC710008 )
  7. 15 ml Falcon tubes (Corning, Falcon®, catalog number: 352099 )
  8. 1.5 ml Eppendorf tubes (USA Scientific, catalog number: 1615-5500 )
  9. 12-well plates (Corning, catalog number: 3513 )
  10. 24-well plates (Corning, Costar®, catalog number: 3526 )
  11. 96-well plates (Greiner Bio One International, catalog number: 650185 )
  12. Sterile 500 ml 0.22 μm filter system (Corning, catalog number: 430758 )
  13. 29 ga. needles attached to 0.5 ml syringe (Terumo Medical Corporation, Elkton, MD, USA)
  14. BALB/c mice (THE JACKSON LABORATORY, catalog number: 000651 )
  15. HEK 293T (ATCC, catalog number: CRL-3216 )
  16. A20 B-cell lymphoma (ATCC, catalog number: TIB-208 )
  17. pCMV-VSVG plasmid (Addgene, catalog number: 8454 )
  18. pCMVΔR8.91 plasmid (Lifescience Market, catalog number: PVT2323 )
  19. pFULGW (Lentiviral luciferase-IRES-GFP plasmid, Available on request)
  20. Lipofectamine 2000 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668027 )
  21. Opti-MEM (Thermo Fisher Scientific, GibcoTM, catalog number: 11058021 )
  22. Dulbecco’s phosphate buffered saline (DPBS) (Corning, catalog number: 21-031-CM )
  23. Trypsin EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  24. Polybrene (Sigma-Aldrich, catalog number: TR-1003-G )
  25. Propidium iodide (Sigma-Aldrich, catalog number: P4864-10ML )
  26. Bright-GloTM Luciferase Assay system (Promega, catalog number: E2610 )
  27. D-Luciferin, potassium salt (Gold Bio, catalog number: LUCK-1G )
  28. Isoflurane; Abbott Laboratories (Abbott Park, Illinois, USA)
  29. Fetal bovine serum (Corning, catalog number: 35-010-CV )
  30. DMEM–high glucose (Sigma-Aldrich, catalog number: D6429-500ML )
  31. L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  32. Gelatin, 2% in H2O, tissue culture grade (Sigma-Aldrich, catalog number: G1393 )
  33. RPMI (Thermo Fisher Scientific, GibcoTM, catalog number: 11875093 )
  34. Penicillin/streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  35. D10 growth media (see Recipes)
  36. 2% gelatin (see Recipes)
  37. D-Luciferin stock solution (see Recipes)

Equipment

  1. Pipettes (Mettler-Toledo, Rainin, catalog numbers: 17008653 , 17008650 , 17008649 ; Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4641070N )
  2. Tissue culture hood
  3. Tissue culture incubator (Eppendorf, New Brunswick, model: Galaxy® 170 S )
  4. Fluorescent inverted microscope with GFP filter (Leica Microsystems, model: Leica DM IL LED )
  5. FACS-Canto flow cytometer (BD Biosciences)
  6. Microcentrifuge (Eppendorf, model: 5424 )
  7. Table-top centrifuge (Eppendorf, model: 5810 )
  8. Ultracentrifuge (Beckman Coulter, model: L8-80M ) equipped with an SW-28 rotor (Beckman Coulter, model: SW 28 )
  9. Balance (VWR, catalog number: 10204-990 )
  10. Xenogen IVIS Imaging System (Perkin Elmer, Hopkinton, MA, USA)
  11. Tabletop Laboratory Animal Anesthesia System (VetEquip, catalog number: 901806 )
  12. Autoclave

Software

  1. Acquisition software (CellQuest, BD Biosciences)
  2. Analysis software (FlowJo v9.3, TreeStar)
  3. Living Image Software (Caliper Life Sciences, Hopkinton, MA, USA)
  4. Graphing software (GraphPad Prism v7.0c, La Jolla, CA, USA)

Procedure

  1. Lentiviral plasmid transfection and virus production
    1. Prepare tissue culture plates by coating them with 2% gelatin (see Recipes). Briefly rinse each 10-cm plate with 10 ml of the 2% gelatin solution and let dry in a hood.
      Note: The same 10 ml of gelatin solution can be reused to coat several plates.
    2. Plate 293T cells on the gelatin-coated tissue culture plates 24 h before transfection at 6-7.5 x 106 cells per 10 cm plate in 10 ml D10 growth media (see Recipes).
      Note: We prepare 4 plates for each lentivirus preparation.
    3. Transfect 293T cells with lentivirus (e.g., pFULGW) and packaging plasmids (pCMV-ΔR8.91, pCMV-VSVG).
      Notes:
      1. Lentiviruses are classified as Biosafety Level 2 (BSL-2) organisms due to their ability to infect primary human cells and experiments need to be conducted in appropriate facilities.
      2. Transfection efficiency can be checked by imaging GFP expression using an inverted fluorescent microscope.

      1. For each plate, prepare 60 μl of Lipofectamine in 1.5 ml Opti-MEM.
      2. For each plate, also prepare 13.3 μg FULGW, 10 μg pCMV-ΔR8.91, and 6.7 μg pCMV-VSVG in 1.5 ml Opti-MEM.
      3. Mix the Lipofectamine and plasmid containing Opti-MEM together and incubate for 25 min at room temperature.
      4. Remove 2 ml of media from each plate (now 8 ml total).
      5. Add 3 ml of the Lipofectamine/plasmid mix to each plate in a dropwise manner, evenly distributing the mix over the plate.
    4. Incubate for 6 h at 37 °C, aspirate the media and add 10 ml of fresh D10.
    5. Harvest SN (the culture media) at 48 h and store at 4 °C. Add another 10 ml fresh D10 media and harvest again at 72 h following transfection. Combine the SNs and clear by centrifugation at 2,000 rpm (805 x g) for 5’ in a table-top centrifuge. Then filter SN through a 0.45 μm filter that has been pre-wetted with 10 ml D10.
    6. Concentrate the virus.
      Note: Use proper containment when working with lentiviruses as per BSL-2 and institutional guidelines.
      1. Ultra-centrifugation method
        1. Put the SN in Beckman ultra-clear 25 x 89 mm tubes (each holds up to 35 ml) and weigh on a balance to the hundredth of a gram to insure they are of equal weight such that the rotor will be balanced.
        2. Spin at 25,000 rpm (112,000 x g) for 90 min at 4 °C in an SW-28 rotor.
        3. Discard supernatant by inverting the tube. Maintain the tube in the inverted position in the tissue culture hood and aspirate the remaining fluid from the walls of the tube to dry it completely without disturbing the viral pellet.
          Note: The pellet will be small and can be difficult to visualize.
        4. Add 200 μl of serum-free media directly to the viral pellet and allow it to resuspend for 12 h at 4 °C. Gently pipette to improve resuspension.
      2. Filtration method
        1. Place SN in Centricon Plus-70 unit (Millipore, Bedford, MA). Each unit holds approximately 60 ml.
        2. Centrifuge at 3,000 rpm (1811 x g) at 15 °C for 2-2.5 h using a table-top centrifuge.
        3. Discard flow through.
        4. Invert unit and spin at 1,500 rpm (453 x g) at 15 °C for 3 min to collect the viral concentrate.
    7. Aliquot 20 μl of virus per Eppendorf tube and store at -80 °C.
      Note: Aliquoting the virus helps to avoid freeze-thaw cycles, each of which will decrease the MOI by about 50%.

  2. Titer lentivirus
    1. One day prior, plate 293T cells in 24-well plates at 250,000 cells/well in 0.5 ml D10.
    2. Transfect cells by adding the equivalent of 10, 3, 1, 0.3, 0.1, and 0 μl of viral concentrate per well of each prep.
    3. After 2 days, harvest cells by rinsing with 100 μl of PBS and then adding 100 μl per well of trypsin-EDTA and incubating for 5 min at 37 °C.
    4. FACS cells and determine % GFP positive.
    5. Calculate:


  3. Cell line transduction
    1. Mix 5-10 MOI of lentivirus in serum-free media with polybrene (6-8 μg/ml) and use it to replace the media of your cell line in a 12- or 24-well tissue culture plate. Spin transfect at 32 °C for 3-4 h at 1,000 x g (~2,300 rpm on a table-top centrifuge).
    2. Incubate at 37 °C for another 3 h, then replace the media with fresh culture media.
    3. Repeat viral transduction the next day if starting with lower MOI.
    4. Grow for 2 days and check transduction efficiency with FACS or with a fluorescent microscope. GFP expression is heterogeneous in cells 2 days following transduction with FULGW and will decrease over time in culture (Figure 2A). 
      Notes:
      1. This decrease likely is due to the competition of untransduced clones or transgene loss where viral integration did not occur.
      2. When testing by FACS, add 0.5 μg/ml of propidium iodide to the FACS solution to exclude dead cells.

  4. Isolate luciferase expressing clones
    1. Plate transduced cells into round-bottom 96-well plates with the goal of obtaining one positive cell per well. The most efficient method to accomplish this is to FACS sort individual GFP-expressing cells 2-3 days after transduction into wells containing 100 μl of growth media. Alternatively, single-cell clones can be obtained by methods of limiting dilution. For example, by preparing plates with 100 μl of growth media containing ~5 cells/ml.
      Note: Plate multiple plates to ensure the development of sufficient clones.
    2. Allow cell clones to grow for ~2 weeks, giving them another 100 μl of fresh culture media at 1 week. Observe for clone growth by simply looking for wells with media that is becoming yellow, or by holding the plate up to a light and looking for colonies in the bottom of the wells.
    3. Transfer the clones to 24-well plates and expand over a few days.
    4. Test for GFP expression by FACS analysis. Clones derived from single cells will have a homogenous, narrow range of GFP expression. Select clones with different levels of expression for further testing (Figure 2B).


      Figure 2. Assessing tumor cell line transduction by FACS. A. GFP expression in A20 cells 2 days and 2 weeks following transduction with FULGW. B. Isolated A20-Luc/GFP clones (#1, 12 and 20) demonstrate stable and homogenous GFP expression.

    5. Test for luciferase activity.
      1. Place 10,000 cells in round-bottom 96-well tissue culture plates.
      2. To each well add a volume of Bright-GloTM Reagent equal to the volume of culture medium in the well, and mix. For 96-well plates, typically 100 μl of reagent is added to cells in 100 μl of culture medium.
      3. Wait at least 2 min to allow cell lysis, then measure luminescence in a luminometer or an IVIS system (Figures 3A and 3B).
    6. Select clones and retest for GFP and luciferase expression after two weeks of culture to insure stable integration of the transgenes.
    7. Freeze and store multiple aliquots of the cell line in liquid nitrogen for future use.


      Figure 3. Confirmation of luciferase expression. A. Imaging A20 and A20-Luc/GFP B-cell lymphoma clones #1, 20, and 12 (10,000 cells from each clone are plated in a round-bottom 96-well plate) and tested for luciferase activity using an IVIS imaging system following the addition of D-Luciferin. B. Luminescence for each clone is quantified in photons per second (p/sec).

  5. Image luciferase cell lines in vivo
    1. It is important to determine whether cell lines will grow efficiently in vivo.
    2. Administer 0.5-1 x 106 Luc/GFP tumors cells either subcutaneously or intravenously into mice of the same genetic background (e.g., BALB/c for A20 cells).
      Note: It may be necessary to precondition mice with 4 Gy of total body irradiation 4 h prior to tumor administration to permit tumor growth in some strains.
    3. Quantify tumor burden by measuring luciferase activity by IVIS (Figure 4).
      1. Inject mice with D-Luciferin (150 mg/kg, i.p.) and anesthetize by exposure to 4% isoflurane in an anesthetic chamber.
      2. Once sedated, transfer the mice onto the pre-warmed stage inside the IVIS imaging system specimen chamber with continuous exposure to 2% isoflurane flowing into the nose-cone to sustain sedation.
      3. Image mice and measure photon flux approximately 10 min following injection of the substrate.


      Figure 4. Monitoring tumor burden in mouse leukemia model. Luminescence imaged by IVIS at 7 and 14 days following, A. subcutaneous (s.c.) injection of 1 x 106 A20-gfp/luc clones on the right flank, or B. after intravenous (i.v.) injection of 5 x 105 A20-gfp/luc clones by tail vein injection.

    4. Euthanize mice when primary tumors reach ~1 cm3 (estimate from palpation), if animals lose significant weight (> 20%), develop hind-limb paralysis or become moribund, whichever comes first according to the method approved by your Institutional Animal Care and Use Committee (IACUC). For intravenously injected leukemia models, where tumor burden is difficult to determine by palpation, we typically euthanize animals when their measured luminescence reaches 107 p/sec.
    5. As a control, obtain a baseline measurement of luminescence in an untreated mouse following D-Luciferin injection. 

Data analysis

IVIS data is collected in regions of interest and can be exported to Excel spreadsheet for analysis. The significance of differences between treatment groups can be determined by unpaired Student’s t-test.

Recipes

  1. D10 growth media
    Add 50 ml of heat-inactivated fetal bovine serum (FBS) to 450 ml DMEM supplemented with glutamine and glucose
  2. 2% gelatin
    Dissolve 1 mg gelatin in 500 ml H2O and autoclave at 121 °C, 15 psi for 30 min
  3. D-Luciferin stock solution
    Dilute to 15 mg/ml in 1x Dulbecco’s phosphate buffered saline (DPBS) (Life Technologies) and sterile-filtered (0.22 μm)

Acknowledgments

  1. This work was supported by grants CA136934 (Y.Y.), CA047741 (Y.Y.), AI083000 (Y.Y.) and AI101263 (T.V.B.) from the National Institutes of Health. Conflict of interest: The authors have declared that no conflict of interest exists.

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简介

鼠肿瘤模型对于我们对肿瘤生理学知识和有效肿瘤治疗方法发展的进展至关重要。 这些研究的关键是能够跟踪肿瘤发展并量化体内肿瘤负荷。 为此,引入赋予肿瘤生物发光特性的基因已经成为啮齿动物肿瘤研究的重要进展。 通过病毒转导引入生物发光基因(例如萤火虫萤光素酶)的方法已经允许产生可以在体内纵向长时间地进行的肿瘤细胞系。 在这里我们描述了通过慢病毒转导产生稳定表达荧光素酶的肿瘤细胞系的方法。

【背景】体内跟踪细胞最重要的是能够通过微创方法从外部检测它们。使用来自萤火虫的荧光素酶(Photinus pyralis )的酶促生物发光是用于体内基于图像的细胞追踪的广泛使用的方法。生物发光已被用于各种体内应用,包括报告基因表达的无创成像(Herschman,2004),研究昼夜节律(Southern and Millar,2005),成像脑卒中(Vandeputte
萤火虫荧光素酶氧化物萤光素在分子氧,镁和三磷酸腺苷存在下在560nm产生黄绿色光(Wilson和Hastings,1998; Fraga,2008)。萤光素酶生物发光在细胞追踪中的益处包括渗透组织用于非侵入性监测以及酶标记物的可再利用性。萤光素酶的另一个优点是大多数细胞不发光,因此可以实现高信噪比。限制性生物发光是由介入的组织例如皮肤,骨头或头发引起的光子衰减。

萤火虫萤光素酶是由luc基因指定的单一多肽,其可以容易地克隆到用于基因递送的载体中。通过质粒转染或非整合病毒转导的瞬时表达限制了可以进行细胞追踪的时间。对于可能持续数月的肿瘤研究来说,这尤其成问题。逆转录病毒整合到基因组中的能力是有利于它们用于产生稳定的细胞系的关键属性。然而,一些肿瘤逆转录病毒,如莫洛尼鼠白血病病毒可能随着时间的推移而受到转基因沉默的限制(Jähner等人,1982)。此外,逆转录病毒载体需要细胞分裂进行基因组整合,并且在转导高度分化的细胞如神经元,树突细胞或静息淋巴细胞时可能效率低下。

为了制备表达荧光素酶的细胞系,来源于人类免疫缺陷病毒-1(HIV-1)的慢病毒逆转录病毒载体非常有效。慢病毒载体优于其他逆转录病毒载体的优点是它们能够整合到非分裂细胞的基因组中。该特性使其成为靶向高度分化细胞(例如神经元,树突细胞和淋巴细胞)的合适的基因递送载体(Naldini等人,1996)。慢病毒还提供非常稳定的基因组整合和长期转基因表达,以至于它们已经用于在胚胎转导后制造转基因小鼠(Lois等人,2002)。

为了追踪体内鼠白血病模型中的血液肿瘤细胞,我们制造了共表达萤火虫萤光素酶(Luc)和增强型绿色荧光蛋白(EGFP)的FULGW慢病毒载体用于B细胞淋巴瘤(A20)细胞系转导和稳定的克隆生产。 FULGW载体基于以前由Miyoshi等人(1998)描述的自灭活载体,该载体已被工程化以表达由人类泛素-C启动子控制的GFP报道基因, et al。(2002),制作FUGW载体。为了制作FULGW,我们用来自Cheadle等人(2010)以前开发的rKat.Luc2.IRES.EGFP的Luc-IRES-EGFP序列替换了FUGW的EGFP序列。
FULGW含有增强基因整合和表达的独特元件(图1A)。它编码人类免疫缺陷病毒-1(HIV-1)皮瓣元件,赋予其非复制性质,允许在非复制细胞中有效的基因组整合(Zennou等,2000)。它含有通过转录稳定增加基因表达的木卡盘肝炎病毒转录后调节元件(WPRE)(Zufferey等人,1999)。此外,它还包括一个3'自我失活的长末端重复序列(3'si-LTR),有助于维持它作为复制缺陷型病毒。通过缺失3'LTR的U3区中的133bp区(ΔU3)使得整合的前病毒的5'LTR转录失活而开发了3'si-LTR(Miyoshi等人, 1998)。

通过用慢病毒质粒(FULGW)和两种包装质粒pCMV-ΔR8.91和pCMV-VSVG(图1B)共转染HEK-293T细胞进行病毒产生。 HEK-293T细胞是稳定表达CMV大T抗原的人胚胎肾细胞系,其大大增加CMV启动子的基因表达,产生强大的病毒产生。 pCMVΔR8.91是HIV-1 Gag和聚合酶(Pol)表达质粒,其通过缺失四种辅助HIV-1基因Vif,Vpr,Vpu和Nef从dR8.9载体修饰(Zufferey等人。,1997)。 pCMV-VSVG表达来源于水泡性口膜炎病毒糖蛋白(VSVG)的泛视包膜(Env)蛋白(Stewart等人,2003)。注:FULGW和pCMV-ΔR8.91都是大质粒,最适合生长在高转化效率的化学感受态recA1缺陷型大肠杆菌中,如生长在30°C的One Shot TOP10大肠杆菌(Invitrogen)持续24-28小时]。使用与这些辅助质粒一起包装的FULGW慢病毒载体,我们已经在各种遗传背景上产生了多种类型的肿瘤细胞系,其稳定表达萤光素酶和GFP以用于肿瘤研究(表1)。


图1. FULGW慢病毒的产生和肿瘤细胞系的转导A.包含Luc-IRES-EGFP转基因的FULGW载体的关键区域的图。转基因表达由人泛素-C启动子驱动。 CMV(巨细胞病毒启动子),U5(LTR独特5'区),R(LTR重复区),HIV-1瓣(人类免疫缺陷病毒-1瓣片元件),Luc(萤火虫萤光素酶),IRES(核糖体内元件序列) ,EGFP(增强型绿色荧光蛋白),WPRE(木卡盘肝炎病毒转录后调节元件),si-LTR(自我失活LTR)。 B.通过用pCMV-ΔR8.91和pCMV-VSVG亲脂共转染包装并假型包装FULGW。在48和72小时收集富含病毒的培养物上清液(SN),并通过超速离心浓缩病毒。在聚凝胺存在下,通过自旋转导将浓缩的病毒用于转导肿瘤细胞系。图解示意图使用Motifolio模板( www.motifolio.com/ )。

表1.通过FULGW转导产生的表达萤光素酶-GFP的肿瘤细胞系

关键字:慢病毒, 肿瘤, 淋巴瘤, 白血病, 荧光素酶, GFP, 小鼠, 方法

材料和试剂

  1. 移液器吸头(USA Scientific,产品目录号:1110-3000,1110-1000,1111-2021)
  2. T75烧瓶(Corning,Falcon ,目录号:353136)
  3. 100毫米TC处理的组织培养皿(Corning,Falcon ,目录号:353003)
  4. 无菌注射器0.45μm过滤器(VWR,目录号:28145-505)
  5. Beckman超清晰25 x 89 mm管(Beckman Coulter,目录号:344060)
  6. Centricon Plus-70单元(Merck,目录号:UFC710008)

  7. 15ml Falcon管(Corning,Falcon ,目录号:352099)

  8. 1.5 ml Eppendorf管(USA Scientific,目录号:1615-5500)
  9. 12孔板(Corning,目录号:3513)
  10. 24孔板(Corning,Costar ®,产品目录号:3526)
  11. 96孔板(Greiner Bio One International,目录号:650185)
  12. 无菌500毫升0.22微米过滤系统(康宁,目录号:430758)
  13. 29 ga。针头连接到0.5毫升注射器(Terumo Medical Corporation,Elkton,MD,USA)
  14. BALB / c小鼠(THE JACKSON LABORATORY,目录号:000651)
  15. HEK 293T(ATCC,目录号:CRL-3216)
  16. A20 B细胞淋巴瘤(ATCC,目录号:TIB-208)
  17. pCMV-VSVG质粒(Addgene,目录号:8454)
  18. pCMVΔR8.91质粒(Lifescience Market,目录号:PVT2323)
  19. pFULGW(慢病毒荧光素酶-IRES-GFP质粒,可根据要求提供)
  20. Lipofectamine 2000(Thermo Fisher Scientific,Invitrogen TM,目录号:11668027)
  21. Opti-MEM(Thermo Fisher Scientific,Gibco TM,目录号:11058021)
  22. 达尔伯克磷酸盐缓冲盐水(DPBS)(Corning,目录号:21-031-CM)
  23. 胰蛋白酶EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25200056)
  24. 聚凝胺(Sigma-Aldrich,目录号:TR-1003-G)
  25. 碘化丙啶(Sigma-Aldrich,目录号:P4864-10ML)
  26. Bright-Glo TM萤光素酶测定系统(Promega,目录号:E2610)
  27. D-萤光素,钾盐(Gold Bio,目录号:LUCK-1G)
  28. 异氟醚;雅培实验室(雅培公园,伊利诺伊州,美国)
  29. 胎牛血清(Corning,目录号:35-010-CV)
  30. DMEM-高葡萄糖(Sigma-Aldrich,目录号:D6429-500ML)
  31. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:25030081)
  32. 明胶,2%H 2 O,组织培养级(Sigma-Aldrich,目录号:G1393)
  33. RPMI(Thermo Fisher Scientific,Gibco TM,目录号:11875093)
  34. 青霉素/链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  35. D10生长介质(见食谱)
  36. 2%明胶(见食谱)
  37. D-Luciferin原液(见食谱)

设备

  1. 移液管(Mettler-Toledo,Rainin,目录号:17008653,17008650,17008649; Thermo Fisher Scientific,Thermo Scientific TM,目录号:4641070N)
  2. 组织培养罩
  3. 组织培养培养箱(Eppendorf,New Brunswick,型号:Galaxy 170S)
  4. 带GFP滤光片的荧光倒置显微镜(Leica Microsystems,型号:Leica DM IL LED)
  5. FACS-Canto流式细胞仪(BD Biosciences)
  6. 微量离心机(Eppendorf,型号:5424)
  7. 台式离心机(Eppendorf,型号:5810)
  8. 装备有SW-28转子(Beckman Coulter,型号:SW 28)的超速离心机(Beckman Coulter,型号:L8-80M)
  9. 余额(VWR,目录号:10204-990)
  10. Xenogen IVIS成像系统(Perkin Elmer,Hopkinton,MA,USA)
  11. 桌面实验室动物麻醉系统(VetEquip,目录号:901806)
  12. 高压灭菌器

软件

  1. 采集软件(CellQuest,BD Biosciences)
  2. 分析软件(FlowJo v9.3,TreeStar)
  3. 生活图像软件(Caliper Life Sciences,Hopkinton,MA,USA)
  4. 绘图软件(GraphPad Prism v7.0c,La Jolla,CA,USA)

程序

  1. 慢病毒质粒转染和病毒生产
    1. 通过用2%明胶包被制备组织培养板(参见食谱)。用10毫升2%明胶溶液简单地冲洗每个10厘米的平板,并在通风橱中干燥。
      注意:同样的10毫升明胶溶液可以重复使用来涂覆几块板。
    2. 在10ml D10生长培养基(参见配方)中以6-7.5×10 6个细胞/ 10cm平板转染前24小时在明胶包被的组织培养板上平板293T细胞。
      注:我们为每种慢病毒制剂准备4块板。
    3. 用慢病毒(例如,pFULGW)和包装质粒(pCMV-ΔR8.91,pCMV-VSVG)转染293T细胞。
      备注:
      1. 由于其能够感染原代人类细胞,因此慢病毒被归类为生物安全级别2(BSL-2)生物体,并且实验需要在适当的设施中进行。
      2. 转染效率可通过使用倒置荧光显微镜对GFP表达进行成像来检查。

      1. 对于每个平板,在1.5ml Opti-MEM中制备60μlLipofectamine。
      2. 对于每个平板,还制备在1.5ml Opti-MEM中的13.3μgFULGW,10μgpCMV-ΔR8.91和6.7μgpCMV-VSVG。
      3. 将Lipofectamine和含有Opti-MEM的质粒混合在一起,并在室温下孵育25分钟。

      4. 每个平板移除2毫升培养基(现在总共8毫升)。
      5. 以逐滴的方式向每个平板添加3ml的Lipofectamine /质粒混合物,将混合物均匀地分布在平板上。

    4. 在37°C孵育6 h,吸出培养基并加入10 ml新鲜D10。
    5. 在48小时收获SN(培养基)并储存在4℃。再加入10ml新鲜的D10培养基并在转染后72小时再次收获。合并SN并通过在台式离心机中以2,000rpm(805×gg)离心5'进行澄清。然后通过0.45μm过滤器过滤SN,过滤器已用10 ml D10预湿。
    6. 集中病毒。
      注意:根据BSL-2和机构指南,在使用慢病毒时应采取适当的遏制措施。
      1. 超离心法
        1. 将SN放入Beckman超清晰25 x 89 mm管(每个容量可达35 ml),并称重至百分之一克,以确保它们具有相同的重量,从而使转子达到平衡。 >
        2. 在SW-28转子中于4℃以25,000rpm(112,000×g克)旋转90分钟。
        3. 倒置管弃去上清液。将管保持在组织培养罩中的倒置位置,并从管壁抽吸剩余的流体以完全干燥,而不会干扰病毒颗粒。
          注意:颗粒很小,很难看见。
        4. 将200μl无血清培养基直接加入到病毒沉淀中,并使其在4°C下重悬12 h。轻轻吸管,以改善重新悬浮。
      2. 过滤方法
        1. 将SN置于Centricon Plus-70单元(Millipore,Bedford,MA)中。
          每个单位约有60毫升

        2. 使用台式离心机在15℃下以3,000rpm(1811gxg)离心2-2.5小时。
        3. 丢弃流量。
        4. 反转装置并在15℃下以1,500rpm(453×g克)旋转3分钟以收集病毒浓缩物。
    7. 每个Eppendorf管分装20μl病毒,并保存在-80°C。
      注意:等分病毒有助于避免冻融循环,每个循环都会使MOI降低约50%。

  2. 滴度慢病毒
    1. 一天前,将24孔板中的293T细胞以250,000个细胞/孔在0.5ml D10中平板培养。
    2. 通过加入相当于每次制备物每孔10,3,1,0.3,0.1和0μl病毒浓缩物的转染细胞。
    3. 2天后,通过用100μlPBS冲洗收集细胞,然后每孔加入100μl胰蛋白酶-EDTA并在37℃孵育5分钟。
    4. FACS细胞并确定%GFP阳性。
    5. 计算:

  3. 细胞系转导
    1. 在无血清培养基中混合5-10 MOI的慢病毒和聚凝胺(6-8μg/ ml),并用它来代替12或24孔组织培养板中的细胞系培养基。以1,000×g(在台式离心机上〜2300rpm)在32℃转染3-4小时。

    2. 在37°C孵育3小时,然后用新鲜培养基更换培养基。

    3. 如果以较低的MOI开始,第二天重复病毒转导。
    4. 培养2天,用FACS或荧光显微镜检查转导效率。在用FULGW转导后2天细胞中GFP表达是不均一的,并且随着培养时间的延长,其表达量会下降(图2A)。
      备注:
      1. 这种下降可能是由于没有病毒整合的未转导克隆或转基因丧失的竞争。
      2. 当通过FACS检测时,向FACS溶液中加入0.5μg/ ml的碘化丙锭以排除死细胞。

  4. 分离萤光素酶表达克隆
    1. 将板转导的细胞放入圆底96孔板中,目标是每孔获得一个阳性细胞。完成此操作的最有效方法是在转导至含有100μl生长培养基的孔中2-3天后,FACS分选单个GFP表达细胞。或者,可以通过有限稀释的方法获得单细胞克隆。例如,通过用含有〜5个细胞/ ml的100μl生长培养基制备平板。
      注意:打印多个印版以确保足够克隆的发展。
    2. 让细胞克隆生长约2周,在1周时再给他们100μl新鲜培养基。通过简单地寻找培养基变黄的孔或通过将培养板保持光线并在孔底寻找菌落观察克隆的生长情况。
    3. 将克隆转移到24孔板并在几天内扩展。
    4. 通过FACS分析测试GFP表达。来自单细胞的克隆将具有均匀,窄范围的GFP表达。选择具有不同表达水平的克隆进行进一步测试(图2B)。


      图2.通过FACS评估肿瘤细胞系转导A.用FULGW转导2天和2周后A20细胞中的GFP表达。 B.分离的A20-Luc / GFP克隆(#1,12和20)表现出稳定和均一的GFP表达。

    5. 测试萤光素酶活性。
      1. 将10,000个细胞放入圆底96孔组织培养板中。
      2. 向每个孔中加入一定体积的Bright-Glo TM试剂,该试剂的量等于孔中培养基的体积,并混合。对于96孔板,通常将100μl试剂加入到100μl培养基中的细胞中。
      3. 等待至少2分钟以使细胞裂解,然后测量发光计或IVIS系统中的发光(图3A和3B)。
    6. 选择克隆并在培养两周后重新测试GFP和萤光素酶表达,以确保转基因稳定整合。
    7. 将多个细胞系的等分试样冷冻并保存在液氮中以备将来使用。


      图3.荧光素酶表达的证实A.成像A20和A20-Luc / GFP B细胞淋巴瘤克隆#1,20和12(来自每个克隆的10,000个细胞在圆底96孔板)中,并在添加D-荧光素之后使用IVIS成像系统测试萤光素酶活性。 B.每个克隆的发光量以每秒光子数(p / sec)定量。

  5. 图像萤光素酶细胞系体内
    1. 确定细胞系是否能在体内高效生长很重要。
    2. 将0.5-1×10 6 Luc / GFP肿瘤细胞皮下或静脉注射到相同遗传背景的小鼠中(例如对于A20细胞,BALB / c)。 /> 注:在肿瘤给药前4小时,可能需要预先照射4 Gy全身照射的小鼠,以允许某些菌株的肿瘤生长。
    3. 通过用IVIS测量萤光素酶活性来量化肿瘤负荷(图4)。
      1. 用D-萤光素(150mg / kg,腹腔注射)注射小鼠并通过在麻醉室中暴露于4%异氟烷麻醉。
      2. 一旦镇静,将小鼠转移到IVIS成像系统样品室内的预热阶段,持续暴露于2%异氟醚流入鼻锥以维持镇静。
      3. 在注射底物后大约10分钟,图像小鼠并测量光子通量。


      图4.监测小鼠白血病模型中的肿瘤负荷在7和14天后通过IVIS成像的发光,A皮下(sc)注射1×10 6个A20- (iv)通过尾静脉注射注射5×10 5个A20-gfp / luc克隆后,在右侧腹腔注射gfp / luc克隆。
    4. 根据以下公式,如果动物体重明显减轻(> 20%),出现后肢瘫痪或濒死,则以原发肿瘤达到〜1 cm3(从触诊估计)安乐死小鼠方法由您的机构动物护理和使用委员会(IACUC)批准。对于通过触诊难以确定肿瘤负荷的静脉内注射的白血病模型,我们通常在测得的发光达到10 7 p / s时安乐死动物。
    5. 作为对照,获得D-Luciferin注射后未处理小鼠中发光的基线测量结果。

数据分析

IVIS数据收集在感兴趣的区域,可以导出到Excel电子表格中进行分析。治疗组之间差异的显着性可以通过不配对的Student's t - 测试来确定。

食谱

  1. D10生长介质
    加入50ml热灭活的胎牛血清(FBS)至补充有谷氨酰胺和葡萄糖的450ml DMEM中。
  2. 2%明胶
    将1mg明胶溶解在500ml H 2 O中并在121℃,15psi下高压灭菌30分钟。
  3. D-Luciferin原液
    在1x Dulbecco's磷酸盐缓冲盐水(DPBS)(Life Technologies)中稀释至15mg / ml并无菌过滤(0.22μm)。

致谢

  1. 这项工作得到了美国国立卫生研究院的赠款CA136934(Y.Y.),CA047741(Y.Y.),AI083000(Y.Y.)和AI101263(T.V.B.)的支持。利益冲突:作者声明不存在利益冲突。

参考

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引用:Brennan, T. V., Lin, L., Huang, X. and Yang, Y. (2018). Generation of Luciferase-expressing Tumor Cell Lines. Bio-protocol 8(8): e2817. DOI: 10.21769/BioProtoc.2817.
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Neeta Bala
Neeta Bala
Hello,
I wanted to request the pFULGW plasmid. Please could you tell me how to proceed?
Thank you,
Neeta Bala
5/30/2018 5:29:01 AM Reply