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Peer-reviewed

Glioma Induction by Intracerebral Retrovirus Injection

脑内注射逆转录病毒诱导胶质瘤

Ravinder K Verma Ravinder K Verma
Fanghui Lu Fanghui Lu
Qing Richard Lu Qing Richard Lu

发布: 2017年07月20日第7卷第14期 DOI: 10.21769/BioProtoc.2404 浏览次数: 9876

评审: Emilie ViennoisKathrin SutterAnonymous reviewer(s)

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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) search Retrovirus (逆转录病毒) search Intracerebral injection (脑内注射) search Murine model (小鼠模型) search PDGFBB (PDGFBB) search PTEN (PTEN) search P53 (P53) search

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

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文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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

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分类

癌症生物学 > 通用技术 > 动物模型

癌症生物学 > 肿瘤免疫学 > 动物模型

细胞生物学 > 组织分析 > 组织分离

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