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Glioma Induction by Intracerebral Retrovirus Injection
脑内注射逆转录病毒诱导胶质瘤

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Abstract

Glioblastoma (GBM) is the most common primary brain cancer in adults and has a poor prognosis. It is characterized by a high degree of cellular infiltration that leads to tumor recurrence, atypical hyperplasia, necrosis, and angiogenesis. Despite aggressive treatment modalities, current therapies are ineffective for GBM. Mouse GBM models not only provide a better understanding in the mechanisms of gliomagenesis, but also facilitate the drug discovery for treating this deadly cancer. A retroviral vector system that expresses PDGFBB (Platelet-derived growth factor BB) and inactivates PTEN (Phosphatase and tensin homolog) and P53 tumor suppressors provides a rapid and efficient induction of glioma in mice with full penetrance. In this protocol, we describe a simple and practical method for inducing GBM formation by retrovirus injection in the murine brain. This system gives a spatial and temporal control over the induction of glioma and allows the assessment of therapeutic effects with a bioluminescent reporter.

Keywords: Glioma(胶质瘤), Retrovirus(逆转录病毒), Intracerebral injection(脑内注射), Murine model(小鼠模型), PDGFBB(PDGFBB), PTEN(PTEN), P53(P53)

Background

Glioblastoma (GBM) is the most aggressive malignant brain tumor and unfortunately is also almost always fatal. Despite advances in multiple therapeutic modalities, no effective therapy has been developed to cure GBM. The mechanisms underlying this disease remain poorly understood. Animal models have been a very important tool to define the GBM pathogenesis and test for gene or drug therapeutics. Several mouse models have been developed with the aim to produce a disease which mimics the human disease as closely as possible and exhibits similar molecular, genetic and histological character. The prominently used models are xenograft (Hingtgen et al., 2008) models where human tumor cell lines can be transplanted orthotopically in brain as well as ectopically in subcutaneous area in immunocompromised mice, providing an advantage of having a large tumor mass in a brief period. Genetically engineered mice models (GEMM) with specific gain-of oncogenic activities or loss of tumor suppressor pathways, which resemble perturbations in the most frequently dysregulated pathways in GBM, results in formation of gliomas in rodents. Genetic perturbations in GEMM models often include the gain-of-function mutations in oncogenic factors such as EGFR, PI-3K, and Ras (Holland et al., 2000; Zhu et al., 2009), and PDGF amplification (Assanah et al., 2006), as well as the loss-of-function in tumor suppressors such as NF1 (Zhu et al., 2005), TP53 (p53) (Zheng et al., 2008), Ink4a/ARF (Holland, 2001), PTEN (Holland et al., 1998 and 2000). The use of viral vectors (Ikawa et al., 2003; Hambardzumyan et al., 2009; Marumoto et al., 2009; Friedmann-Morvinski et al., 2012) such as retrovirus, lentivirus and adenovirus carrying oncogenes and/or targeting tumor suppressors has been a convenient and effective approach to induce brain tumor formation in rodents. There is a need for testing multiple different tumor models for therapeutics since different subtypes of gliomas have varied genetic profiles and clinical responses to drug treatment.
   GBM have been classified into four subtypes depending upon their gene expression pattern and molecular signature, namely, Mesenchymal, Neural, Proneural and Classical tumors (Verhaak et al., 2010). The Proneural subtype of GBM predominantly involves mutations/loss in oncogene P53 and amplification of PDGFRα with loss of PTEN seen across all the subtypes (Verhaak et al., 2010; Lei et al., 2011). To closely imitate the human Proneural subtype, Lei et al. (2011) devised a GBM model by inducing PTEN and P53 deletion and PDGFBB overexpression in the progenitor cells of the white matter region of adult murine brain through retrovirus inoculation (Lei et al., 2011). We have successfully used this retrovirus-induced GBM model in mice ranging in age from day 2 to 3-month-old adult mice with remarkable reproducibility (Lu et al., 2016). This model develops gliomas with full penetrance within 3-4 weeks which in contrast to other GEMM models, which take a significantly longer time.

Materials and Reagents

  1. Materials needed for culture and virus packaging
    1. Culture dishes 10 cm diameter (Corning, Falcon®, catalog number: 353003 )
    2. 0.45 µm size filter (Corning® bottle-top vacuum filter system 500 ml) (Corning, catalog number: 430512 )
    3. 38 ml open-end tubes (Beckman Coulter, catalog number: 344058 )
    4. 50 ml screw capped tubes (Corning, Falcon®, catalog number: 352098 )
    5. 15 ml screw capped conical tubes (Corning, Falcon®, catalog number: 352196 )
    6. Round bottom 5 ml tubes (Beckman Coulter, catalog number: 344057 )
    7. Parafilm
    8. Vector system
      1. Retroviral vector carrying PDGFB-IRES-Cre
      2. Retroviral packaging plasmid pEco (Clontech, catalog number: PT3749-5 )

        Note: We use a two-vector system for retrovirus production. One is a retroviral vector carrying PDGFB-IRES-Cre, which overexpresses PDGFB and Cre, and another is a retroviral packaging plasmid, which provides gag, pol and env to produce VSV-G pseudotyped retrovirus. We use commercially available DNA transfection reagents for 293T HEK transfection to produce the retrovirus.

    9. Low passaged 293T HEK cells (P6-P14)
    10. Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11960044 )
    11. Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11550 )
    12. L-glutamate (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
    13. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
    14. Sodium pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11360070 )
    15. Trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
    16. Transfection agent: Polyjet (SignaGen Laboratories, catalog number: SL100688 )
    17. 20% sucrose in HBSS (Hank’s Balanced Salt Solution) (Thermo Fisher Scientific, GibcoTM, catalog number: 14025134 )
    18. Complete media for 293T cells (D10 culture media) (see Recipes)

  2. Materials needed for titration of retrovirus
    1. 24-well plate
    2. Isolated MEF cells (105 cells/well)
    3. CAG-Rosa-tdTomato reporter mice (THE JACKSON LABORATORY, catalog number: 007909 )
    4. Concentrated virus
    5. Poly-L-lysine (Sigma-Aldrich, catalog number: P7890 ), 0.05 mg/ml prepared in sterile tissue-culture grade water, filter sterilized
    6. Sterile tissue culture grade water (Sigma-Aldrich, catalog number: W3500 )
    7. Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11960044 )
    8. Fetal bovine serum (FBS) (Atlas Biologicals, catalog number: S11550 )
    9. L-glutamate (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
    10. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
    11. Sodium pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11360070 )
    12. Complete media for MEF cells (D10 culture media) (see Recipes)

  3. Materials needed for intracranial stereotaxic injection
    1. 1 cc syringes (BD, catalog number: 309659 ) and 30 G needles (BD, catalog number: 305128 )
    2. Autoclaved cotton tips
    3. Animals
      Ptenfl/fl (THE JACKSON LABORATORY, catalog number: 006440 )
      Trp53fl/fl (THE JACKSON LABORATORY, catalog number: 008462 )
      Notes:
      1. These mouse stains were crossed with a reporter line Rosa-tmLuciferase (THE JACKSON LABORATORY, catalog number: 005125 ) which has a firefly luciferase gene inserted at the ROSA26 locus (Durkin et al., 2013). This bioluminescent reporter will help to track the tumor growth and evaluate candidate therapeutic regimens. The mice were maintained on a mixed C57Bl/6; 129Sv; CD-1 background.
      2. Obtain appropriate animal use protocol from Institutional Animal Care and Use Committee (IACUC) before the animal work.
    4. Polybrene (1 µg/ml) (Sigma-Aldrich, catalog number: 107689 )
    5. Isoflurane (Piramal®) for inhalational anesthesia (2-3% for induction), or Intraperitoneal Route: ketamine (Ketaset®, NDC 0856-2013-01) + xylazine (Anased® 20 mg/ml, Santa Cruz Biotechnology, catalog number: sc-362950Rx ) combination
    6. Buprenorphine (Buprenex® 0.3 mg/ml): Dilution 1 ml of Buprenex + 9.0 ml D5W for use in mice
    7. Sterile phosphate buffered saline
    8. 70% ethanol
    9. Chlorhexidine (BactoShield® CHG 2% Surgical Scrub) (STERIS, catalog number: 132224 )
    10. Isopropanol (Priority Care®, Isopropyl Alcohol 70%) (FIRST PRIORITY, catalog number: MS070PC )
    11. GLUture® Topical Tissue Adhesive (Abbott, catalog number: 32046-04-01 )
    12. Artificial Tears Lubricant Ophthalmic Ointment (Henry Schein, catalog number: 18581 )
    13. Ketamine and xylazine combination (see Recipes)

Equipment

  1. Equipment needed for ultracentrifugation concentration
    1. Pipettes
    2. Sterile working hood
    3. Ultracentrifuge (Beckman Coulter, model: Optima XPN-90 or equivalent)
    4. Shaker
    5. Rotors:
      1. SW 32 Ti (Beckman Coulter, catalog number: 369650 )
      2. SW 55 Ti (Beckman Coulter, catalog number: 342196 )

  2. Equipment needed for titration of retrovirus
    1. Biosafety hood
    2. Neubauer cell counting chamber

  3. Equipment needed for surgery
    1. Stereotaxic device (RWD Life Science, catalog number: 68502 with mouse adapter: RWD Life Science, catalog number: 68010 )
    2. Micro-drill (RWD Life Science, catalog number: 78001 ) to make a burr hole in the skull with appropriate drill bits (RWD Life Science, catalog number: 78002 )
    3. Autoclaved surgical pack composed of scissors, forceps, cotton swabs and tips, applicators, scalpel blade holder
    4. 10 μl Hamilton syringe (Hamilton, catalog number: 7659-01 ) with 30 G blunt-end needle (Hamilton, catalog number: 7803-07 )
    5. Incubator or heating pad
    6. Timer
    7. Biobubble or sterile working place
    8. Surgical site Marker (MEDTRONIC, DevonTM, catalog number: 31145793 )
    9. Anesthesia Machine (Ohmeda, model: Excel 210SE or equivalent)

Procedure


Figure 1. Schematic of the whole procedure. A. Viral packaging and concentration; B. Stereotaxic intracranial injection of virus for induction of glioma.

  1. Transfection and viral packaging procedure (Figure 1A)
    Note: We follow the manufacturer provided protocol for Polyjet mediated transfection (http://signagen.com/DataSheet/SL100688.pdf).
    1. Day 1: Plating of 293T cells
      Plate 293T cells (around 3-4 x 106) in poly-L-lysine coated 10 cm diameter dishes in DMEM with 10% FBS media (D10 culture media). Cells will become 70-80% confluent after 24 h.
      Note: Plan accordingly to allow 5-6 h next day for transfection and medium change.
    2. Day 2: Transfection day
      1. Pre-transfection check
        1. Check for confluence next day and use the plates with 70-80% cell confluence.
          Note: Higher confluence can be a hindrance as cells don’t have space to grow.
        2. 1 h before transfection, change media with fresh D10 media–4 ml per plate.
        3. Use the following table for 10 cm plates for transfection (Table 1).

          Table 1. Transfection mix


      2. Transfection
        1. Mix the calculated concentration of DNA in 500 µl DMEM without FBS and gently mix.
        2. Add 45 µl of Polyjet in 455 µl of serum free DMEM media and gently mix with pipette and immediately mix with diluted DNA of step A2b i. Gently mix them and incubate at room temperature for 10-15 min.
          Note: Not more than 20 min.
        3. Then add it into the plates drop wise and gently shake.
        4. Incubate at 37 °C and 5% CO2 for 5 h.
          Note: Do not keep longer as it can be toxic to the cells.
        5. Discard the transfecting media and add fresh D10 10 ml per plate and incubate at 37 °C and 5% CO2 for 24 h.
    3. Day 3: 1st collection
      1. Collect the media and centrifuge at 600 x g (2,000 rpm) for 10 min and collect supernatant, leaving the cellular debris at the bottom. Collected media can be stored at 4 °C for 3 days at max (Tiscornia et al., 2006) before ultracentrifuge concentration.
      2. Add fresh D10 media 10 ml per plate and incubate again for next 24 h.
    4. Day 4: 2nd collection
      1. Collect the media and centrifuge at 600 x g (2,000 rpm) for 10 min and collect the supernatant, leaving the cellular debris at the bottom.
      2. Pool the media from day 3 and day 4 and filter using 0.45 µm pore size filter.
    5. Ultracentrifugation for viral concentration
      1. We use Beckman Coulter rotor SW 32 Ti, which has capacity to hold six 38 ml open-end tubes. Balance the tubes to the maximum difference of 0.5 g and centrifuge at 68,300 x g (20,000 rpm) for 2 h at 4 °C. With each tube holding 30 ml, a total 360 ml of media can be centrifuged in one run. If required to run more media, discard the supernatant after 1st spin without disturbing the pellet add fresh media for 2nd run.
        Note: Do not use the tubes filled less than half of its capacity which increases the risk of collapse.
      2. Pour off the supernatant after the run and aspirate rest of the droplets from the wall without disturbing the pellet.
      3. Transfer the centrifuge tubes in to 50 ml screw capped tubes and keep them upright on shaker at 4 °C overnight and allow the residual media to re-suspend pellet (roughly around 20-30 μl/tube).
      4. Centrifuge briefly and collect the re-suspended pellets from all the tubes, aliquot the concentrate in 4-10 μl volumes and store at -80 °C for more than month. Avoid repeated cycles of thawing and freezing that can drastically reduce the viral titer.
      5. For in vivo injection, further purification of virus by sucrose cushion is recommended (Tiscornia et al., 2006).
    6. Sucrose cushion
      1. Collect the viral concentrate after overnight shaking and wash the tube with 100 µl of HBSS and pool it with collected viral concentrate.
      2. Using round bottom 5 ml tubes, fill it with 1.5 ml of 20% sucrose in HBSS and add 200 μl of viral concentrate. The rotor used is SW 55 Ti and after balancing, spin at 48,600 x g (20,000 rpm) for 2 h at 4 °C.
      3. Pour off the supernatant and allow rest to drain off by inverting the tubes. Re-suspend the pellet in 50-100 μl of HBSS, cover with Parafilm and shake slowly for 20-30 min.
      4. Collect the concentrate, aliquot it and store at -80 °C.
      5. Check for the titer in a reporter cell line. We use mouse embryonal fibroblasts, which express tdTomato reporter upon Cre expression.

  2. Titration of retrovirus
    MEF (mouse embryonic fibroblasts) cells are isolated from E13.5-14.5 embryos of CAG-Rosa-tdTomato reporter mice (THE JACKSON LABORATORY) (Durkin et al., 2013) for retrovirus titering. These cells express tdTomato when exposed to Cre-expressing viruses and allowing us to assess the viral infectivity. For virus titration, we use the procedure described by Tiscornia et al. (2006).
    1. Coat the 24-well plate with 500 µl poly-L-lysine per well with incubation at 37 °C for 30 min, aspirate the media and wash with autoclaved deionized water and let it dry for 1-2 h in biosafety hood.
    2. Plate 105 MEF cells in 500 µl D10 media per well in 24-well plate. Use minimum of 8 wells including one as negative control.
    3. Incubate it for 6 h and then check after 6 h if they are adhered to the plate.
    4. Make 10-fold dilutions of retrovirus (undiluted to 106) in PBS with total volume around 50 µl.
    5. Change 480 µl D10 media with polybrene at a concentration of 4 µg/ml.
    6. Add 20 µl of each viral dilution to the well and mix gently and thoroughly.
    7. Incubate the cells at 37 °C for 48 h. TdTomato will be visible within 24-48 h.
    8. Assess the percentage of tdTomato positive cells by counting on Neubauer cell counting chamber or by using FACS and calculate the titer by using following formula:



      Viral titer ≥ 107/ml is adequate for tumor formation.

  3. Intracranial stereotaxic injection procedure (Figure 1B)
    Note: Neonatal or young adult mice can also be used with appropriate stereotaxic device adapter.
    1. Pre-surgical preparation
      1. Switch on the incubator or heating pad.
      2. Check the micro-drill, anesthesia machine for oxygen pressure and adequate isoflurane flow and overhead light source.
      3. Set up the stereotaxic instrument as per the instructions of the manufacturer.
      4. Switch on the anesthesia machine with attachment for mouse enclosure. Oxygen flow (0.8-1.5 L/min) and isoflurane (2-3%) are set for induction of anesthesia and decreased to 1-1.5% for maintenance. Alternatively use ketamine and xylazine combination which is injected intra-peritoneal.
      5. Load the concentrated virus in syringe (e.g., 3 µl per each mouse to be injected) slowly avoiding bubbles and set it up on the holder in stereotaxic device.
      6. Apply artificial tear ointment on exposed cornea to avoid drying.
    2. Surgical procedure
      1. Anesthetize the mouse and check the anesthesia level by toe pinch.
      2. Set up the mice on stereotaxic device with nose and ear holders firmly to avoid any accessory movement of head during the procedure and isolate the surgical area with sterile drape or covers.
      3. Clean the operative area (back of the skull) using autoclaved cotton tips with chlorhexidine and isopropanol alternatively three times each. Wait for a few minutes.
      4. Make an incision on back of head of about 1-1.5 cm in length to expose bregma.
      5. Adjust the stereotaxic device in relation to bregma as a reference and then use the coordinates depending on the age and area of brain to be targeted (e.g., for targeting the corpus callosum in adult mice, we used the coordinates: Anteroposterior + 0.2 mm, Lateral ± 0.5 mm and dorso-ventral 2.2 mm.
      6. Mark the targeted area with surgical site marker.
      7. Drill hole in bone using micro-drill being careful to avoid going too deep.
      8. Lower the syringe needle 0.4-0.6 mm deeper to the desired depth (e.g., here 2.2 + 0.6 mm), wait for 1-2 min and retract back to desired depth to form a small pocket and slowly inject 1 μl of viral concentrate, wait for 1-2 min, retract 1 mm and inject 1 μl again. We usually inject 1-3 μl of viral concentrate in one adult mouse.
      9. Slowly retract the needle to avoid leaking of the concentrate.
      10. After complete retraction, use tissue glue to join the tissue flaps.
      11. Give buprenorphine injection intra-peritoneal. (0.05-0.1 mg/kg)
      12. Keep the mouse on heating pad or incubator till it becomes conscious.
      13. Monitor the mice for next 2-3 days and administer analgesics as per the protocol.
      14. Wash the needle with 70% ethanol and three times with sterile water.

Data analysis

The initial tumor growth can be noticed within 2-3 weeks in the brain monitored by in vivo bioluminescent imaging system such as IVIS Spectrum CT (PerkinElmer). Tumor-bearing mice normally survive for 3-6 weeks post injection depending on age at the time of injection. Neonatal mice injected at age postnatal day 2 (P2) to day 4 (P4) (48-96 h) tend to survive longer and develop a larger tumor, possibly due to the expansion of non-fused calvarial bones. The brain sections are stained with hematoxylin and eosin and tumors observed are typically invasive and hypercellular with hyperchromatic nuclei, foci of palisading necrosis and microvascular proliferation suggestive of glioblastoma (Figure 2).


Figure 2. H&E histology of tumor sections. A. Sections showing tumor growth in the brain (Dotted line enclosure); B. Section showing typical features of glioblastoma-palisading necrosis (yellow arrow) and microvascular proliferation (blue arrowheads). Scale bars in (A) 1 mm and (B) 50 µm.

Notes

  1. Use only healthy and low passaged 293T or 293FT cells for virus packaging.
  2. Do not use filter smaller than 0.45 µm for the risk of shearing effect on viral particles.

Recipes

  1. Complete media for 293T and MEF cells (D10 culture media)
    435 ml DMEM medium supplemented with 50 ml FBS
    5 ml L-glutamine
    5 ml penicillin-streptomycin
    5 ml sodium pyruvate
  2. Ketamine and xylazine combination
    1 ml ketamine (Ketaset® 100 mg/ml)
    0.5 ml of xylazine (Anased® 20 mg/ml)
    8.5 ml of D5W (5% dextrose in water)

Acknowledgments

This work was supported in part by grants from the US National Institutes of Health NS078092 and R37 NS096359 to QRL.

References

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简介

成胶质细胞瘤(GBM)是成人中最常见的原发性脑癌,预后差。其特征在于高度的细胞浸润,导致肿瘤复发,非典型增生,坏死和血管生成。尽管采取积极的治疗方式,目前的疗法对GBM无效。小鼠GBM模型不仅提供了对胶质瘤发生机制的更好理解,而且有助于药物发现治疗这种致命的癌症。表达PDGFBB(血小板衍生生长因子BB)和灭活PTEN(磷酸酶和张力蛋白同源物)和P53肿瘤抑制因子的逆转录病毒载体系统提供了具有全面外显子的小鼠中胶质瘤的快速和有效的诱导。在该方案中,我们描述了一种简单实用的方法,用于在鼠脑中通过逆转录病毒注射诱导GBM形成。该系统对神经胶质瘤的诱导进行空间和时间控制,并允许用生物发光报告物评估治疗效果。
【背景】胶质母细胞瘤(GBM)是最具侵袭性的恶性脑肿瘤,不幸的是几乎总是致命的。尽管多种治疗方式有进展,但是尚未开发出治疗GBM的有效治疗方法。这种疾病的机制仍然知之甚少。动物模型一直是定义GBM发病机制和基因或药物治疗试验的非常重要的工具。已经开发了几种小鼠模型,其目的是产生尽可能接近模拟人类疾病的疾病,并表现出类似的分子,遗传和组织学特征。突出使用的模型是异种移植(Hingtgen等人,2008)模型,其中人类肿瘤细胞系可以在脑中原位移植以及在免疫受损的小鼠的皮下区域异位移植,提供了具有大肿瘤块在短时间内。遗传工程小鼠模型(GEMM)具有特异性增益的致癌活性或丧失肿瘤抑制通路,其类似于GBM中最频繁失调的通路中的扰动,导致啮齿类动物形成胶质瘤。 GEMM模型中的基因扰动通常包括致癌因子如EGFR,PI-3K和Ras中的功能增益突变(Holland等人,2000; Zhu等人, ,2009)和PDGF扩增(Assanah等人,2006),以及肿瘤抑制因子如NF1的功能丧失(Zhu et al。 o,2005),TP53(p53)(Zheng等人,2008),Ink4a / ARF(Holland,2001),PTEN(Holland等人, 1998年和2000年)。使用病毒载体(Ikawa等人,2003; Hambardzumyan等人,2009; Marumoto等人,2009; Friedmann- Morvinski等人,2012),如逆转录病毒,慢病毒和携带致癌基因和/或靶向肿瘤抑制因子的腺病毒已经成为诱导啮齿动物脑瘤形成的一种方便有效的方法。需要测试多种不同的肿瘤模型用于治疗,因为不同的胶质瘤亚型具有不同的遗传概况和药物治疗的临床反应。
 根据其基因表达模式和分子标记,即间充质,神经,自主和古典肿瘤(Verhaak等人,2010),GBM已被分为四种亚型。 GBM的Proneural亚型主要涉及致癌基因P53的突变丧失和PDGFRα的扩增,所有亚型均可见PTEN的丧失(Verhaak等人,2010; Lei等人,,2011)。为了密切模仿人类Proneural亚型,Lei等人(2011)通过诱导PTEN 和P53 删除和设计了一种GBM模型, PDGFBB通过逆转录病毒接种在成年鼠脑白质区域的祖细胞中过度表达(Lei等人,2011)。我们已经成功地将这种逆转录病毒诱导的GBM模型用于具有显着重现性的第2至3月龄成年小鼠年龄的小鼠(Lu等人,2016)。该模型在3-4周内开发具有完全外显率的胶质瘤,与其他GEMM模型相比,其具有显着更长的时间。

关键字:胶质瘤, 逆转录病毒, 脑内注射, 小鼠模型, PDGFBB, PTEN, P53

材料和试剂

  1. 培养和病毒包装所需的材料
    1. 直径10厘米的培养皿(Corning,Falcon ®,目录号:353003)
    2. 0.45微米尺寸的过滤器(Corning,/)真空过滤系统500毫升)(康宁,目录号:430512)
    3. 38毫升开口管(Beckman Coulter,目录号:344058)
    4. 50ml螺旋盖管(Corning,Falcon ®,目录号:352098)
    5. 15毫升螺旋锥形管(Corning,Falcon ®,目录号:352196)
    6. 圆底5毫升管(Beckman Coulter,目录号:344057)
    7. 石蜡膜
    8. 矢量系统
      1. 携带PDGFB-IRES-Cre的逆转录病毒载体
      2. 逆转录病毒包装质粒pEco(Clontech,目录号:PT3749-5)

        注意:我们使用双向载体系统进行逆转录病毒生产。一种是携带PDGFB-IRES-Cre的逆转录病毒载体,其过表达PDGFB和Cre,另一种是逆转录病毒包装质粒,其提供gag,pol和env产生VSV-G假型逆转录病毒。我们使用市售的DNA转染试剂进行293T HEK转染以产生逆转录病毒

    9. 低通293T HEK细胞(P6-P14)
    10. Dulbecco改性Eagle培养基(DMEM)(Thermo Fisher Scientific,Gibco TM,目录号:11960044)
    11. 胎牛血清(FBS)(Atlanta Biologicals,目录号:S11550)
    12. L-谷氨酸(Thermo Fisher Scientific,Gibco TM,目录号:25030081)
    13. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
    14. 丙酮酸钠(Thermo Fisher Scientific,Gibco TM,目录号:11360070)
    15. 胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25300054)
    16. 转染剂:Polyjet(SignaGen Laboratories,目录号:SL100688)
    17. HBSS中的20%蔗糖(Hank's Balanced Salt Solution)(Thermo Fisher Scientific,Gibco TM,目录号:14025134)
    18. 293T细胞(D10培养基)的完整培养基(参见食谱)

  2. 逆转录病毒滴定所需的材料
    1. 24孔板
    2. 分离的MEF细胞(10 细胞/孔)
    3. CAG-Rosa-tdTomato记者小鼠(The JACKSON LABORATORY,目录号:007909)
    4. 集中病毒
    5. 将聚-L-赖氨酸(Sigma-Aldrich,目录号:P7890),在无菌组织培养级水中制备的0.05mg / ml,过滤灭菌的
    6. 无菌组织培养级水(Sigma-Aldrich,目录号:W3500)
    7. Dulbecco改性Eagle培养基(DMEM)(Thermo Fisher Scientific,Gibco TM,目录号:11960044)
    8. 胎牛血清(FBS)(Atlas Biologicals,目录号:S11550)
    9. L-谷氨酸(Thermo Fisher Scientific,Gibco TM,目录号:25030081)
    10. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
    11. 丙酮酸钠(Thermo Fisher Scientific,Gibco TM,目录号:11360070)
    12. 用于MEF细胞(D10培养基)的完整培养基(参见食谱)

  3. 颅内立体定位注射所需材料
    1. 1 cc注射器(BD,目录号:309659)和30 G针(BD,目录号:305128)
    2. 高压灭菌棉提示
    3. 动物
      (JACKSON LABORATORY,目录号:006440)
      (JACKSON LABORATORY,目录号:008462)
      注意:
      1. 将这些小鼠斑点与在ROSA26基因座插入的萤火虫荧光素酶基因(Durkin等人,2013)的报道系Rosa-tmLuciferase(THE JACKSON LABORATORY,目录号:005125)杂交。该生物发光记者将有助于跟踪肿瘤生长并评估候选治疗方案。将小鼠保持在混合的C57B1 / 6; 129SV; CD-1背景。
      2. 在动物工作之前,从机构动物护理和使用委员会(IACUC)获得适当的动物使用协议。
    4. 聚凝胺(1μg/ ml)(Sigma-Aldrich,目录号:107689)
    5. 用于吸入麻醉的异氟烷(Piramal )(2-3%用于诱导)或腹膜内途径:氯胺酮(Ketaset ,NDC 0856-2013-01)+甲苯噻嗪圣诞老人克鲁兹生物技术公司,目录号:sc-362950Rx)组合
    6. 丁丙诺啡(Buprenex 0.3 mg / ml):稀释1 ml Buprenex + 9.0 ml D5W,用于小鼠
    7. 无菌磷酸盐缓冲盐水
    8. 70%乙醇
    9. 氯己定(BactoShield CHG 2%手术洗刷)(STERIS,目录号:132224)
    10. 异丙醇(优先保养,异丙醇70%)(FIRST PRIORITY,目录号:MS070PC)
    11. GLUture ®局部组织粘合剂(Abbott,目录号:32046-04-01)
    12. 人造泪液润滑剂眼科软膏(Henry Schein,目录号:18581)
    13. 氯胺酮和赛拉嗪组合(见食谱)

设备

  1. 超离心浓缩所需的设备
    1. 移液器
    2. 无菌工作罩
    3. 超速离心机(Beckman Coulter,型号:Optima XPN-90或同等级)
    4. 振动器
    5. 转子:
      1. SW 32 Ti(Beckman Coulter,目录号:369650)
      2. SW 55 Ti(Beckman Coulter,目录号:342196)

  2. 滴定逆转录病毒所需的设备
    1. 生物安全罩
    2. Neubauer细胞计数室

  3. 手术所需的设备
    1. 立体定位装置(RWD Life Science,目录号:68502,带有鼠标适配器:RWD Life Science,目录号:68010)
    2. 微钻(RWD Life Science,目录号:78001),用适当的钻头在头骨上制作一个钻孔(RWD Life Science,目录号:78002)
    3. 高压灭菌手术包由剪刀,镊子,棉签和尖端组成,敷贴器,手术刀刀片架
    4. 10μlHamilton注射器(Hamilton,目录号:7659-01),带有30G平头针(Hamilton,目录号:7803-07)
    5. 孵化器或加热垫
    6. 计时器
    7. 生物泡或无菌工作地点
    8. 手术部位标记(MEDTRONIC,Devon TM ,目录号:31145793)
    9. 麻醉机(Ohmeda,型号:Excel 210SE或同等学历)

程序


图1.整个程序的示意图。 A.病毒包装和浓缩; B.立体定位颅内注射病毒以诱导胶质瘤。

  1. 转染和病毒包装程序(图1A)
    注意:我们遵循制造商提供的Polyjet介导转染方案( http://signagen.com/DataSheet/SL100688.pdf )。
    1. 第1天:电镀293T细胞
      在具有10%FBS培养基(D10培养基)的DMEM中的聚-L-赖氨酸包被的10cm直径的培养皿中的板293T细胞(约3-4×10 6)。细胞在24小时后将达到70-80%汇合。
      注意:相应地计划允许第二天5-6小时进行转染和中等变化。
    2. 第2天:转染日
      1. 预转染检查
        1. 检查第二天的汇合,并使用70-80%细胞汇合的板。
          注意:更高的汇合可能是一个障碍,因为细胞没有生长空间。
        2. 转染前1 h,用新鲜的D10培养基更换培养基,每片4ml
        3. 使用下表进行10cm平板转染(表1)
          表1.转染组合


      2. 转染
        1. 将计算出的DNA浓度在500μl不含FBS的DMEM中混合,并轻轻混合
        2. 在455μl无血清DMEM培养基中加入45μlPolyjet,并用移液管轻轻混合,并立即与步骤A2b i的稀释DNA混合。轻轻混匀,室温孵育10-15分钟。
          注意:不超过20分钟。
        3. 然后将其加入板中轻轻晃动。
        4. 在37℃和5%CO 2下孵育5小时。
          注意:不要保持更长时间,因为它可能对细胞有毒。
        5. 弃去转染培养基,加入新鲜的D10 10 ml每个板,37℃和5%CO 2孵育24 h。
    3. 第3天:第1集
      1. 收集介质并以600 x g(2000 rpm)离心10分钟,收集上清液,留下细胞碎片在底部。在超速离心浓缩之前,收集的培养基可以在4℃下储存最多3天(Tiscornia等人,2006)。
      2. 加入新鲜的D10培养基,每个板10毫升,再孵育24小时。
    4. 第4天:第2集
      1. 收集介质并以600 x g(2000 rpm)离心10分钟,收集上清液,留下细胞碎片在底部。
      2. 从第3天和第4天装载介质,并使用0.45μm孔径的过滤器进行过滤
    5. 超速离心用于病毒浓度
      1. 我们使用Beckman Coulter转子SW 32 Ti,它具有容纳6个38毫升开口管的能力。将管平衡至0.5g的最大差异,并在6℃下以68,300×g(20,000rpm)离心2小时。每个管保持30毫升,总共360毫升的介质可以一次离心。如果需要运行更多的培养基,第一次旋转后丢弃上清液,而不会干扰沉淀物添加新鲜培养基进行第二次运行。
        注意:不要使用充满不到其容量的一半的管道,这会增加崩溃的风险。
      2. 运行后倒出上清液,从墙壁吸出剩余的液滴,不会干扰沉淀物。
      3. 将离心管转移到50ml螺旋封口的管中,并将其在4℃的振荡器上直立放置过夜,并使剩余的介质重新悬浮沉淀(大约在20-30μl/管)。
      4. 短暂离心并收集所有管中的再悬浮颗粒,将浓缩物等分至4-10μl体积,并在-80℃下储存多个月。避免重复的解冻和冻结循环,可以大大降低病毒滴度
      5. 对于体内注射,推荐通过蔗糖垫进一步纯化病毒(Tiscornia等人,2006)。
    6. 蔗糖垫
      1. 收集病毒浓缩物过夜摇匀后,用100μlHBSS洗涤管,并用收集的病毒浓缩液将其收集
      2. 使用圆底5 ml管,在HBSS中加入1.5 ml 20%蔗糖,加入200μl病毒浓缩液。使用的转子是SW 55 Ti,平衡后,在4℃下以48,600×g(20,000rpm)旋转2小时。
      3. 倒出上清液,并通过翻转管子使休息排出。将沉淀重新悬浮在50-100μlHBSS中,用Parafilm盖住,缓慢摇匀20-30分钟。
      4. 收集浓缩物,等分并储存在-80°C。
      5. 检查记者细胞系中的滴度。我们使用小鼠胚胎成纤维细胞,其在Cre表达时表达tdTomato记录
  2. 滴定逆转录病毒
    用于逆转录病毒滴定的CAG-Rosa-tdTomato报告小鼠(The JACKSON LABORATORY)(Durkin等,2013)的E13.5-14.5胚胎分离MEF(小鼠胚胎成纤维细胞)细胞。当暴露于Cre表达病毒时,这些细胞表达tdTomato,并允许我们评估病毒感染性。对于病毒滴定,我们使用Tiscornia等人描述的程序(2006)。
    1. 每孔用500μl聚-L-赖氨酸覆盖24孔板,37℃温育30分钟,吸出培养基,用高压灭菌去离子水洗涤,并在生物安全罩中干燥1-2小时。 />
    2. 在50孔平板中,每孔500μlD10培养基中的平板10μL/孔。最少使用8口井,其中1口为阴性对照
    3. 孵育6小时,然后6小时后检查,如果它们粘在板上。
    4. 在PBS中将10倍稀释的逆转录病毒(未稀释至10×6)稀释至总体积约50μl。
    5. 用聚凝胺改变480μlD10培养基,浓度为4μg/ ml
    6. 将20μl每种病毒稀释液加入孔中,轻轻充分混匀。
    7. 在37℃孵育细胞48小时。 TdTomato将在24-48小时内看到。
    8. 通过计数Neubauer细胞计数室或使用FACS评估tdTomato阳性细胞的百分比,并使用以下公式计算滴度:



      病毒滴度≥10 / ml适合肿瘤形成。

  3. 颅内立体定位注射程序(图1B)
    注意:新生儿或年轻成年小鼠也可以使用适当的立体定位装置适配器。
    1. 手术前准备
      1. 打开孵化器或加热垫。
      2. 检查微钻,麻醉机的氧气压力和足够的异氟醚流量和头顶光源
      3. 按照制造商的说明设置立体定位仪器。
      4. 打开麻醉机附带鼠标外壳。设定氧气流量(0.8-1.5L / min)和异氟烷(2-3%),用于诱导麻醉,维持时减少至1-1.5%。或者使用注射腹膜内的氯胺酮和赛拉嗪组合。
      5. 将浓缩的病毒装入注射器(例如,,每个要注射的小鼠3μl)缓慢避免气泡并将其置于立体定位装置的支架上。
      6. 在暴露的角膜上涂抹人造泪液以避免干燥。
    2. 外科手术
      1. 麻醉老鼠,并通过脚趾捏捏检查麻醉水平。
      2. 在立体定位装置上,用鼻子和耳柄牢固地设置小鼠,以避免在手术过程中头部的任何附件移动,并用无菌悬垂或盖子隔离外科手术区域。
      3. 使用高压灭菌的棉尖用氯己定和异丙醇清洗手术区域(颅骨背部),每次三次。等待几分钟。
      4. 在长度约1-1.5厘米的头后面做一个切口,以暴露出阳。。
      5. 调整相对于母体的立体定位装置作为参考,然后根据大脑的年龄和面积(例如)使用坐标,用于在成年小鼠中靶向胼the体,我们使用坐标:前后+ 0.2mm,横向±0.5mm,背腹2.2mm
      6. 用手术部位标记标记目标区域。
      7. 使用微型钻头在骨头钻孔,小心避免过深。
      8. 将注射器针头深度更低到所需深度(例如,这里为2.2 + 0.6mm),等待1-2分钟,并缩回到所需的深度以形成小口袋,并缓慢注射1μl病毒浓缩液,等待1-2 min,收缩1 mm,再次注入1μl。我们通常在一只成年小鼠中注射1-3μl病毒浓度。
      9. 缓慢收缩针头,以避免浓缩液泄漏。
      10. 完全收缩后,使用组织胶粘合组织瓣。
      11. 给予丁丙诺啡注射腹膜内。 (0.05-0.1 mg / kg)
      12. 将鼠标放在加热垫或培养箱上,直到变得清醒。
      13. 监测小鼠接下来2-3天,并按照方案进行镇痛药。
      14. 用70%乙醇洗涤针头,用无菌水洗涤3次

数据分析

在体内生物发光成像系统如IVIS Spectrum CT(PerkinElmer)监测的大脑中,2-3周内可以注意到初始肿瘤生长。携带肿瘤的小鼠通常在注射后存活3-6周,这取决于注射时的年龄。在出生后第2天(P2)至第4天(P4)(48-96小时)注射的新生儿小鼠倾向于存活较长时间,并且可能由于未融合的颅骨骨骼的扩张而形成较大的肿瘤。脑切片用苏木精和伊红染色,观察到的肿瘤通常是浸润性和超细胞性,具有多色细胞核,栅栏坏死灶和暗示胶质母细胞瘤的微血管增生(图2)。


图2.肿瘤切片的H& E组织学。 A.显示脑中肿瘤生长的部分(虚线外壳); B.显示胶质母细胞瘤斑块坏死(黄色箭头)和微血管增生(蓝色箭头)的典型特征的部分。 (A)中的刻度尺为1mm,(B)为50μm。

笔记

  1. 只能使用健康和低通的293T或293FT细胞进行病毒包装。
  2. 不要使用小于0.45μm的过滤器来对病毒颗粒进行剪切效应。

食谱

  1. 293T和MEF细胞(D10培养基)的完整培养基
    435ml补充有50ml FBS的DMEM培养基 5 ml L-谷氨酰胺
    5 ml青霉素 - 链霉素
    5毫升丙酮酸钠
  2. 氯胺酮和甲噻嗪组合
    1ml氯胺酮(Ketaset 100mg / ml)
    0.5ml的甲苯噻嗪(Anase®20mg / ml)
    8.5ml D5W(5%葡萄糖水溶液)

致谢

这项工作部分得到了美国国家卫生研究院NS078092和R37 NS096359给QRL的资助。

参考

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引用:Verma, R. K., Lu, F. and Lu, Q. (2017). Glioma Induction by Intracerebral Retrovirus Injection. Bio-protocol 7(14): e2404. DOI: 10.21769/BioProtoc.2404.
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