Materials and Reagents

Pipette tips (USA Scientific, catalog numbers: 1110-3000 , 1110-1000 , 1111-2021 )
T75 flask (Corning, Falcon^®, catalog number: 353136 )
100 mm TC-treated Tissue Culture Dish (Corning, Falcon^®, catalog number: 353003 )
Sterile syringe 0.45 μm filter (VWR, catalog number: 28145-505 )
Beckman ultra-clear 25 x 89 mm tubes (Beckman Coulter, catalog number: 344060 )
Centricon Plus-70 unit (Merck, catalog number: UFC710008 )
15 ml Falcon tubes (Corning, Falcon^®, catalog number: 352099 )
1.5 ml Eppendorf tubes (USA Scientific, catalog number: 1615-5500 )
12-well plates (Corning, catalog number: 3513 )
24-well plates (Corning, Costar^®, catalog number: 3526 )
96-well plates (Greiner Bio One International, catalog number: 650185 )
Sterile 500 ml 0.22 μm filter system (Corning, catalog number: 430758 )
29 ga. needles attached to 0.5 ml syringe (Terumo Medical Corporation, Elkton, MD, USA)
BALB/c mice (THE JACKSON LABORATORY, catalog number: 000651 )
HEK 293T (ATCC, catalog number: CRL-3216 )
A20 B-cell lymphoma (ATCC, catalog number: TIB-208 )
pCMV-VSVG plasmid (Addgene, catalog number: 8454 )
pCMVΔR8.91 plasmid (Lifescience Market, catalog number: PVT2323 )
pFULGW (Lentiviral luciferase-IRES-GFP plasmid, Available on request)
Lipofectamine 2000 (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 11668027 )
Opti-MEM (Thermo Fisher Scientific, Gibco^TM, catalog number: 11058021 )
Dulbecco’s phosphate buffered saline (DPBS) (Corning, catalog number: 21-031-CM )
Trypsin EDTA (Thermo Fisher Scientific, Gibco^TM, catalog number: 25200056 )
Polybrene (Sigma-Aldrich, catalog number: TR-1003-G )
Propidium iodide (Sigma-Aldrich, catalog number: P4864-10ML )
Bright-Glo^TM Luciferase Assay system (Promega, catalog number: E2610 )
D-Luciferin, potassium salt (Gold Bio, catalog number: LUCK-1G )
Isoflurane; Abbott Laboratories (Abbott Park, Illinois, USA)
Fetal bovine serum (Corning, catalog number: 35-010-CV )
DMEM–high glucose (Sigma-Aldrich, catalog number: D6429-500ML )
L-Glutamine (Thermo Fisher Scientific, Gibco^TM, catalog number: 25030081 )
Gelatin, 2% in H₂O, tissue culture grade (Sigma-Aldrich, catalog number: G1393 )
RPMI (Thermo Fisher Scientific, Gibco^TM, catalog number: 11875093 )
Penicillin/streptomycin (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
D10 growth media (see Recipes)
2% gelatin (see Recipes)
D-Luciferin stock solution (see Recipes)

Equipment

Pipettes (Mettler-Toledo, Rainin, catalog numbers: 17008653 , 17008650 , 17008649 ; Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 4641070N )
Tissue culture hood
Tissue culture incubator (Eppendorf, New Brunswick, model: Galaxy^® 170 S )
Fluorescent inverted microscope with GFP filter (Leica Microsystems, model: Leica DM IL LED )
FACS-Canto flow cytometer (BD Biosciences)
Microcentrifuge (Eppendorf, model: 5424 )
Table-top centrifuge (Eppendorf, model: 5810 )
Ultracentrifuge (Beckman Coulter, model: L8-80M ) equipped with an SW-28 rotor (Beckman Coulter, model: SW 28 )
Balance (VWR, catalog number: 10204-990 )
Xenogen IVIS Imaging System (Perkin Elmer, Hopkinton, MA, USA)
Tabletop Laboratory Animal Anesthesia System (VetEquip, catalog number: 901806 )
Autoclave

Software

Acquisition software (CellQuest, BD Biosciences)
Analysis software (FlowJo v9.3, TreeStar)
Living Image Software (Caliper Life Sciences, Hopkinton, MA, USA)
Graphing software (GraphPad Prism v7.0c, La Jolla, CA, USA)

Procedure

Lentiviral plasmid transfection and virus production
1. Prepare tissue culture plates by coating them with 2% gelatin (see Recipes). Briefly rinse each 10-cm plate with 10 ml of the 2% gelatin solution and let dry in a hood.
  Note: The same 10 ml of gelatin solution can be reused to coat several plates.
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 (see Recipes).
  Note: We prepare 4 plates for each lentivirus preparation.
3. Transfect 293T cells with lentivirus (e.g., pFULGW) and packaging plasmids (pCMV-ΔR8.91, pCMV-VSVG).
  Notes:
  1. Lentiviruses are classified as Biosafety Level 2 (BSL-2) organisms due to their ability to infect primary human cells and experiments need to be conducted in appropriate facilities.
  2. Transfection efficiency can be checked by imaging GFP expression using an inverted fluorescent microscope.
  1. For each plate, prepare 60 μl of Lipofectamine in 1.5 ml Opti-MEM.
  2. For each plate, also prepare 13.3 μg FULGW, 10 μg pCMV-ΔR8.91, and 6.7 μg pCMV-VSVG in 1.5 ml Opti-MEM.
  3. Mix the Lipofectamine and plasmid containing Opti-MEM together and incubate for 25 min at room temperature.
  4. Remove 2 ml of media from each plate (now 8 ml total).
  5. Add 3 ml of the Lipofectamine/plasmid mix to each plate in a dropwise manner, evenly distributing the mix over the plate.
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.
  Note: Use proper containment when working with lentiviruses as per BSL-2 and institutional guidelines.
  1. Ultra-centrifugation method
    1. Put the SN in Beckman ultra-clear 25 x 89 mm tubes (each holds up to 35 ml) and weigh on a balance to the hundredth of a gram to insure they are of equal weight such that the rotor will be balanced.
    2. Spin at 25,000 rpm (112,000 x g) for 90 min at 4 °C in an SW-28 rotor.
    3. Discard supernatant by inverting the tube. Maintain the tube in the inverted position in the tissue culture hood and aspirate the remaining fluid from the walls of the tube to dry it completely without disturbing the viral pellet.
      Note: The pellet will be small and can be difficult to visualize.
    4. Add 200 μl of serum-free media directly to the viral pellet and allow it to resuspend for 12 h at 4 °C. Gently pipette to improve resuspension.
  2. Filtration method
    1. Place SN in Centricon Plus-70 unit (Millipore, Bedford, MA). Each unit holds approximately 60 ml.
    2. Centrifuge at 3,000 rpm (1811 x g) at 15 °C for 2-2.5 h using a table-top centrifuge.
    3. Discard flow through.
    4. Invert unit and spin at 1,500 rpm (453 x g) at 15 °C for 3 min to collect the viral concentrate.
7. Aliquot 20 μl of virus per Eppendorf tube and store at -80 °C.
  Note: Aliquoting the virus helps to avoid freeze-thaw cycles, each of which will decrease the MOI by about 50%.
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.
5. Calculate:
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 transfect 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 (Figure 2A).
  Notes:
  1. This decrease likely is due to the competition of untransduced clones or transgene loss where viral integration did not occur.
  2. When testing by FACS, add 0.5 μg/ml of propidium iodide to the FACS solution to exclude dead cells.
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.
  Note: Plate multiple plates to ensure the development of sufficient clones.
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).
  
  Figure 2. Assessing tumor cell line transduction by FACS. A. GFP expression in A20 cells 2 days and 2 weeks following transduction with FULGW. B. Isolated A20-Luc/GFP clones (#1, 12 and 20) demonstrate stable and homogenous GFP expression.
5. Test for luciferase activity.
  1. Place 10,000 cells in round-bottom 96-well tissue culture plates.
  2. To each well add a volume of Bright-Glo^TM Reagent equal to the volume of culture medium in the well, and mix. For 96-well plates, typically 100 μl of reagent is added to cells in 100 μl of culture medium.
  3. Wait at least 2 min to allow cell lysis, then measure luminescence in a luminometer or an IVIS system (Figures 3A and 3B).
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.
  
  Figure 3. Confirmation of luciferase expression. A. Imaging A20 and A20-Luc/GFP B-cell lymphoma clones #1, 20, and 12 (10,000 cells from each clone are plated in a round-bottom 96-well plate) and tested for luciferase activity using an IVIS imaging system following the addition of D-Luciferin. B. Luminescence for each clone is quantified in photons per second (p/sec).
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).
  Note: It may be necessary to precondition mice with 4 Gy of total body irradiation 4 h prior to tumor administration to permit tumor growth in some strains.
3. Quantify tumor burden by measuring luciferase activity by IVIS (Figure 4).
  1. Inject mice with D-Luciferin (150 mg/kg, i.p.) and anesthetize by exposure to 4% isoflurane in an anesthetic chamber.
  2. Once sedated, transfer the mice onto the pre-warmed stage inside the IVIS imaging system specimen chamber with continuous exposure to 2% isoflurane flowing into the nose-cone to sustain sedation.
  3. Image mice and measure photon flux approximately 10 min following injection of the substrate.
  Figure 4. Monitoring tumor burden in mouse leukemia model. Luminescence imaged by IVIS at 7 and 14 days following, A. subcutaneous (s.c.) injection of 1 x 10⁶ A20-gfp/luc clones on the right flank, or B. after intravenous (i.v.) injection of 5 x 10⁵ A20-gfp/luc clones by tail vein injection.
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.

Data analysis

IVIS data is collected in regions of interest and can be exported to Excel spreadsheet for analysis. The significance of differences between treatment groups can be determined by unpaired Student’s t-test.

Recipes

D10 growth media
Add 50 ml of heat-inactivated fetal bovine serum (FBS) to 450 ml DMEM supplemented with glutamine and glucose
2% gelatin
Dissolve 1 mg gelatin in 500 ml H₂O and autoclave at 121 °C, 15 psi for 30 min
D-Luciferin stock solution
Dilute to 15 mg/ml in 1x Dulbecco’s phosphate buffered saline (DPBS) (Life Technologies) and sterile-filtered (0.22 μm)

Acknowledgments

This work was supported by grants CA136934 (Y.Y.), CA047741 (Y.Y.), AI083000 (Y.Y.) and AI101263 (T.V.B.) from the National Institutes of Health. Conflict of interest: The authors have declared that no conflict of interest exists.

References

Brennan, T. V., Lin, L., Brandstadter, J. D., Rendell, V. R., Dredge, K., Huang, X. and Yang, Y. (2016). Heparan sulfate mimetic PG545-mediated antilymphoma effects require TLR9-dependent NK cell activation. J Clin Invest 126(1): 207-219.
Byrne, F. L., McCarroll, J. A. and Kavallaris, M. (2016). Analyses of tumor burden in vivo and metastasis ex vivo using luciferase-expressing cancer cells in an orthotopic mouse model of neuroblastoma. Methods Mol Biol 1372: 61-77.
Cheadle, E. J., Hawkins, R. E., Batha, H., O'Neill, A. L., Dovedi, S. J. and Gilham, D. E. (2010). Natural expression of the CD19 antigen impacts the long-term engraftment but not antitumor activity of CD19-specific engineered T cells. J Immunol 184(4): 1885-1896.
Costa, G. L., Sandora, M. R., Nakajima, A., Nguyen, E. V., Taylor-Edwards, C., Slavin, A. J., Contag, C. H., Fathman, C. G. and Benson, J. M. (2001). Adoptive immunotherapy of experimental autoimmune encephalomyelitis via T cell delivery of the IL-12 p40 subunit. J Immunol 167(4): 2379-2387.
Fraga, H. (2008). Firefly luminescence: a historical perspective and recent developments. Photochem Photobiol Sci 7(2): 146-158.
Herschman, H. R. (2004). Noninvasive imaging of reporter gene expression in living subjects. Adv Cancer Res 92: 29-80.
Jähner, D., Stuhlmann, H., Stewart, C. L., Harbers, K., Lohler, J., Simon, I. and Jaenisch, R. (1982). De novo methylation and expression of retroviral genomes during mouse embryogenesis. Nature 298(5875): 623-628.
Jenkins, D. E., Hornig, Y. S., Oei, Y., Dusich, J. and Purchio, T. (2005). Bioluminescent human breast cancer cell lines that permit rapid and sensitive in vivo detection of mammary tumors and multiple metastases in immune deficient mice. Breast Cancer Res 7(4): R444-454.
Lois, C., Hong, E. J., Pease, S., Brown, E. J. and Baltimore, D. (2002). Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295(5556): 868-872.
Miyoshi, H., Blomer, U., Takahashi, M., Gage, F. H. and Verma, I. M. (1998). Development of a self-inactivating lentivirus vector. J Virol 72(10): 8150-8157.
Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F. H., Verma, I. M. and Trono, D. (1996). In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272(5259): 263-267.
Rosol, T. J., Tannehill-Gregg, S. H., LeRoy, B. E., Mandl, S. and Contag, C. H. (2003). Animal models of bone metastasis. Cancer 97(3 Suppl): 748-757.
Simmons, J. K., Hildreth, B. E., 3rd, Supsavhad, W., Elshafae, S. M., Hassan, B. B., Dirksen, W. P., Toribio, R. E. and Rosol, T. J. (2015). Animal models of bone metastasis. Vet Pathol 52(5): 827-841.
Southern, M. M. and Millar, A. J. (2005). Circadian genetics in the model higher plant, Arabidopsis thaliana. Methods Enzymol 393: 23-35.
Stewart, S. A., Dykxhoorn, D. M., Palliser, D., Mizuno, H., Yu, E. Y., An, D. S., Sabatini, D. M., Chen, I. S., Hahn, W. C., Sharp, P. A., Weinberg, R. A. and Novina, C. D. (2003). Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA 9(4): 493-501.
Vandeputte, C., Reumers, V., Aelvoet, S. A., Thiry, I., De Swaef, S., Van den Haute, C., Pascual-Brazo, J., Farr, T. D., Vande Velde, G., Hoehn, M., Himmelreich, U., Van Laere, K., Debyser, Z., Gijsbers, R. and Baekelandt, V. (2014). Bioluminescence imaging of stroke-induced endogenous neural stem cell response. Neurobiol Dis 69: 144-155.
Wilson, T. and Hastings, J. W. (1998). Bioluminescence. Annu Rev Cell Dev Biol 14: 197-230.
Zennou, V., Petit, C., Guetard, D., Nerhbass, U., Montagnier, L. and Charneau, P. (2000). HIV-1 genome nuclear import is mediated by a central DNA flap. Cell 101(2): 173-185.
Zufferey, R., Donello, J. E., Trono, D. and Hope, T. J. (1999). Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol 73(4): 2886-2892.
Zufferey, R., Nagy, D., Mandel, R. J., Naldini, L. and Trono, D. (1997). Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 15(9): 871-875.