Materials and Reagents

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
9. Low passaged 293T HEK cells (P6-P14)
10. Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, Gibco^TM, catalog number: 11960044 )
11. Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11550 )
12. L-glutamate (Thermo Fisher Scientific, Gibco^TM, catalog number: 25030081 )
13. Penicillin-streptomycin (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
14. Sodium pyruvate (Thermo Fisher Scientific, Gibco^TM, catalog number: 11360070 )
15. Trypsin-EDTA (Thermo Fisher Scientific, Gibco^TM, 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, Gibco^TM, catalog number: 14025134 )
18. Complete media for 293T cells (D10 culture media) (see Recipes)
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, Gibco^TM, catalog number: 11960044 )
8. Fetal bovine serum (FBS) (Atlas Biologicals, catalog number: S11550 )
9. L-glutamate (Thermo Fisher Scientific, Gibco^TM, catalog number: 25030081 )
10. Penicillin-streptomycin (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
11. Sodium pyruvate (Thermo Fisher Scientific, Gibco^TM, catalog number: 11360070 )
12. Complete media for MEF cells (D10 culture media) (see Recipes)
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
  Pten^fl/fl (THE JACKSON LABORATORY, catalog number: 006440 )
  Trp53^fl/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

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:
Equipment needed for titration of retrovirus
1. Biosafety hood
2. Neubauer cell counting chamber
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, Devon^TM, 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.

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
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 a 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
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 a minimum of 8 wells including one as negative control.
3. Incubate it for 6 h and then check after 6 h if they adhere 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.
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

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

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

Recipes

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
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.

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