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Generation of Luciferase-Expressing Tumor Cell Lines Protocol

Experiment Summary

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.

Fig. 1 Production of FULGW lentivirus and transduction of tumor cell lines.Fig. 1 Production of FULGW lentivirus and transduction of tumor cell lines.

Materials and Reagents

  1. Pipette tips
  2. T75 flask
  3. 100 mm TC-treated Tissue Culture Dish
  4. Sterile syringe 0.45 μm filter
  5. Ultra-clear 25 x 89 mm tubes
  6. Centricon Plus-70 unit
  7. 15 ml tubes, 1.5 ml tubes
  8. 12-well plates, 24-well plates, 96-well plates
  9. Sterile 500 ml 0.22 μm filter system
  10. 29 ga. needles attached to 0.5 ml syringe
  11. BALB/c mice
  12. HEK 293T
  13. A20 B-cell lymphoma
  14. pCMV-VSVG plasmid
  15. pCMVΔR8.91 plasmid
  16. pFULGW (Lentiviral luciferase-IRES-GFP plasmid)
  17. Lipofectamine 2000
  18. Opti-MEM
  19. Phosphate buffered saline (DPBS)
  20. Trypsin EDTA
  21. Polybrene
  22. Propidium iodide
  23. Bright-GloTM Luciferase Assay system
  24. D-Luciferin, potassium salt
  25. Isoflurane; Abbott Laboratories
  26. Fetal bovine serum
  27. DMEM–high glucose
  28. L-Glutamine
  29. Gelatin, 2% in H2O, tissue culture grade
  30. RPMI
  31. Penicillin/streptomycin
  32. D10 growth media
  33. 2% gelatin
  34. D-Luciferin stock solution

Equipment

  1. Pipettes
  2. Tissue culture hood
  3. Tissue culture incubator
  4. Fluorescent inverted microscope with GFP filter
  5. FACS-Canto flow cytometer
  6. Microcentrifuge, Table-top centrifuge, Ultracentrifuge
  7. Balance
  8. Xenogen IVIS Imaging System
  9. Tabletop Laboratory Animal Anesthesia System
  10. Autoclave

Procedure

A. Lentiviral plasmid transfection and virus production

1. Prepare tissue culture plates by coating them with 2% gelatin. Briefly rinse each 10-cm plate with 10 ml of the 2% gelatin solution and let dry in a hood.

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.

3. Transfect 293T cells with lentivirus (e.g., pFULGW) and packaging plasmids (pCMV-ΔR8.91, pCMV-VSVG).

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.

7. Aliquot 20 μl of virus per tube and store at -80 °C.

B. 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.

Calculate:

Calculate

C. 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 transfects 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.

D. 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.

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).

Fig. 2 Assessing tumor cell line transduction by FACS.Fig. 2 Assessing tumor cell line transduction by FACS.

5. Test for luciferase activity.

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.

E. 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).

3. Quantify tumor burden by measuring luciferase activity by IVIS (Figure 3).

Figure 3. Monitoring tumor burden in mouse leukemia model.Figure 3. Monitoring tumor burden in mouse leukemia model.

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.

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