Experiment Summary

Glioblastoma (GBM) is the most common primary brain cancer in adults and has a poor prognosis. 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.

Materials and Reagents

A. Materials needed for culture and virus packaging

1. Culture dishes 10 cm diameter
2. 0.45 µm size filter
3. 38 ml open-end tubes, 50 ml screw capped tubes, 15 ml screw capped conical tubes, round bottom 5 ml tubes
4. Vector system (Retroviral vector carrying PDGFB-IRES-Cre, Retroviral packaging plasmid pEco)
5. Low passaged 293T HEK cells
6. Dulbecco’s modified Eagle medium (DMEM)
7. Fetal bovine serum (FBS), L-glutamate, Penicillin-streptomycin, Sodium pyruvate, Trypsin-EDTA, Transfection agent: Polyjet, 20% sucrose in HBSS

B. Materials needed for titration of retrovirus

1. 24-well plate
2. Isolated MEF cells (10⁵ cells/well)
3. CAG-Rosa-tdTomato reporter mice
4. Concentrated virus
5. Poly-L-lysine
6. Sterile tissue culture grade water
7. Complete media for MEF cells

C. Materials needed for intracranial stereotaxic injection

1. cc syringes
2. Autoclaved cotton tips
3. Mice
4. Polybrene (1 µg/ml), Isoflurane, Buprenorphine, Isopropanol
5. Sterile phosphate buffered saline
6. GLUture® Topical Tissue Adhesive
7. Artificial Tears Lubricant Ophthalmic Ointment

Equipment

A. Equipment needed for ultracentrifugation concentration

1. Pipettes
2. Sterile working hood
3. Ultracentrifuge
4. Shaker
5. Rotors: SW 32 Ti, SW 55 Ti

B. Equipment needed for titration of retrovirus

1. Biosafety hood
2. Neubauer cell counting chamber

C. Equipment needed for surgery

1. Stereotaxic device
2. Micro-drill
3. Autoclaved surgical pack composed of scissors, forceps, cotton swabs and tips, applicators, scalpel blade holder
4. 10 μl Hamilton syringe
5. Incubator or heating pad
6. Timer
7. Biobubble or sterile working place
8. Surgical site Marker
9. Anesthesia Machine

Procedure

Fig. 1 Schematic of the whole procedure.

A. Transfection and viral packaging procedure

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.

2. Day 2: Transfection day

1) Pre-transfection check

Check for confluence next day and use the plates with 70-80% cell confluence.

1 h before transfection, change media with fresh D10 media-4 ml per plate.

Use the following table for 10 cm plates for transfection.

2) Transfection

Mix the calculated concentration of DNA in 500 µl DMEM without FBS and gently mix.

Add 45 µl of Polyjet in 455 µl of serum free DMEM media and gently mix with pipette and immediately mix with diluted DNA. Gently mix them and incubate at room temperature for 10-15 min.

Then add it into the plates drop wise and gently shake.

Incubate at 37 °C and 5% CO₂ for 5 h.

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 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) 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.

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.

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.

B. Titration of retrovirus

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.

C. Intracranial stereotaxic injection procedure (Figure 1B)

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.

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 the 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).