Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

Ethics Statement: Every effort is made to keep animal discomfort or stress to a minimum during irradiation and tail vein injections. Mice are used as a source of cells in these experiments; as such there are no procedures or manipulations apart from euthanasia. Mice will be euthanized by CO₂ inhalation followed by cervical dislocation to confirm death. This procedure is consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association.

1. Prepare Retroviral Construct

1. Sub-clone the T-cell receptor (TCR) alpha and beta chains, separated by a 2A linker, into a MSCV-based retroviral¹¹ vector (example, pMIAII or pMIGII)¹¹.
   NOTE: The 2A linker allows for stoichiometric and concordant expression of the alpha and beta TCR chains from a single open reading frame. The vector includes an IRES fluorescent protein cassette (ametrine or GFP) that is used to track transduced cells and transduction efficiency. For a detailed protocol please see Holst et al.¹¹
2. Isolate Plasmid DNA using commercially obtained plasma prep kit or a similar product. Use a working concentration of 1 – 2 μg μl^-1.

2. Generation of Retroviral Producer Cell Lines

1. Utilize a two-step process to produce the retroviral producer cell lines.
   NOTE: For a detailed protocol, please view Holst et al.¹¹.
   1. Generate retrovirus by transient transfection of 293T cells with three plasmids (pVSVG, pEQ-Pam3(-E) and vector of interest¹¹) and take the retroviral supernatant from the 293T cells to transduce the GP+E86 ecotropic retroviral producer cell line.
   2. Isolate the top 40% of fluorescent expressing GP+E86 producer cells by FACS and propagate the transdcuced-GP+E86 producer cells as a source of virus.
      NOTE: The generation of high-titer producers is critical for efficient transduction of bone marrow cells. For a complete protocol please view Holst et al.¹¹

3. Retrovirus-mediated Stem Cell Gene Transfer (Day -5) 

1. Thaw out and culture 0.5 -1.0 x 10⁶ of each GP+E86 producer cell line in complete DMEM 10% (vol/vol) FBS to allow sufficient growth for two confluent 150-mm plates by Day 0 of this protocol.
   NOTE: Thawing 0.5 – 1.0 x 10⁶ of each GP+E86 producer cell line in a 10 cm plate will allow for 30 – 50% confluency at Day 5. This also allows for 5 days of culture in order to expand the GP+E86 producer cell lines to two confluent 150-mm plates by Day 0.
   1. Culture retroviral producer cell line in complete tissue culture media containing 5% feta calf serum, and ciprofloxacin (10 μg/ml) to avoid mycoplasma contamination.
      NOTE: Discontinue use of ciprofloxacin when generating the viral supernatant for bone marrow transduction. Avoid over-growth of the producer cell line, as this will negatively impact bone marrow transduction efficiency.

4. Inject Donor Mice with 5-Fluorouracil (5-FU) (Day -3)

1. Weigh the donor mice (5 – 12 weeks old) to determine the amount of 5-FU required. Using mice younger than 5 weeks of age results in low bone marrow yield. To ensure expression of a single TCR, use a T cell deficient mouse strain such as SCID, RAG or TCR alpha deficient mice as bone marrow donors.
2. Working in the tissue culture hood, dilute the 5-FU stock with sterile PBS in order to make a sufficient amount of 10 mg ml^-1 working solution of 5-FU to deliver 0.15 mg 5-FU per gram of body weight.
3. Using a 1 ml syringe with a 37 G needle, intraperitoneal inject 0.15 mg 5-FU per gram of body weight per donor mouse.
   NOTE: Use 1 donor mouse per one recipient to start and adjust the number of donor mice according to the cell yield. An alternative method to the 5-FU treatment for the enrichment and isolation of bone marrow progenitors is negative selection of lineage positive cells as described in Viret et al.

5. Bone Marrow Extraction Procedure (Day 0)

1. Obtain dissection scissors and forceps for bone isolation. Prepare media (HBSS supplemented with 5% FCS (vol/vol)) to collect bones in a 50 ml conical tube. Keep 50 ml conical containing media on ice. Euthanize mice by CO₂ inhalation followed by cervical dislocation to confirm death. Pinch paw to ensure no reflexes are detected.
2. Sterilize the surgical site and surgical tools with ethanol or methanol. Remove the femur, tibia, humerus, and pelvic girdle carefully by first cutting up and under the pelvic girdle. Cut back down along the femur to the tibia. Remove all surrounding tissue using scissors and forceps to obtain clean bones. Keep bones hydrated in HBSS + 5% (vol/vol) FBS.
3. Separate the bones by cutting at the joints, making sure to cut both ends off each bone. Insert a 25-gauge needle attached to a 10-ml syringe containing HBSS + 5% (vol/vol) FBS. Apply pressure and flush all bone marrow through a 70-μm strainer resting in a 50 ml conical.
4. Periodically, push the bone marrow through the 70-μm filter using the plunger from a 1 ml or 3 ml syringe. Rinse the strainer with HBSS + 5% (vol/vol) FBS. This will ensure a single cell suspension that is free of bone fragments and tissue. Keep cells sterile by performing the procedure in a sterile hood.
5. Centrifuge bone marrow cells at 300 x g for 10 min at 4 °C.

6. Bone Marrow Culture (Day 0)

1. Decant or aspirate media and resuspend cell pellet in 1 ml ammonium chloride based red blood lysis buffer (or RBC lysis buffer equivalent) per 50 ml conical tube. After 1 min incubation at room temperature, quench the red cell lysis buffer by adding 10 ml of complete DMEM 20% (vol/vol) FBS.
2. Centrifuge cells at 300 x g for 10 min at 4 °C.
3. Decant or aspirate supernatant and resuspend bone marrow in 5 ml complete DMEM 20% (vol/vol) FBS. Count the cells with a Neubauer counting chamber.
   NOTE: General yield should be approximately 5 – 10 x 10⁶ cells per mouse; however, the yield can vary among different strains of mice.
4. Plate bone marrow cells at a density of 4 x 10⁷ cells per 150-mm plate in 30 ml of complete DMEM 20% (vol/vol) FBS supplemented with IL-3 (20 g ml^-1), IL-6 (50 g ml^-1) and SCF (50 g ml^-1).
   NOTE: Use freshly thawed-aliquoted cytokines. Do not use cytokines that have been frozen and thawed more than once or kept at 4 °C for more than 2 weeks.
5. Incubate bone marrow overnight (approximately 24 hr) at 37 °C with 5% CO₂.

7. Retroviral Producer Cell Culture (Day 0)

1. Plate 3 x 10⁶ retroviral producer cells per 150-mm plate in 18 ml complete DMEM 20% (vol/vol) FBS.
2. Incubate retroviral producer cells at 37 °C overnight. Anticipate one 150-mm plate for 15 million bone marrow cells (approximately 2 – 3 donor mice).

8. Retroviral Supernatant (Day 1)

1. The next day (approximately 24 hr later), harvest the retroviral supernatant from the producer plates (set up in step 7) by drawing up the retroviral supernatant directly off of the 150-mm plate with a 10-ml syringe and filter using a 0.45-μm syringe filter.
2. Carefully replace the removed retroviral supernatant with 18 ml of fresh complete DMEM 20% (vol/vol) FBS.
3. Supplement the filtered retroviral supernatant with IL-3 (20 g ml^-1), IL-6 (50 g ml^-1), MSCF (50 g ml^-1) and Hexadimethrine bromide (Polybrene) (6 μg ml^-1).
   NOTE: Hexadimethrine bromide should be made fresh every 2 months to avoid a drop in efficacy of retroviral transduction.

9. Bone Marrow Harvest (Day 1)

1. After completing step 8.3, harvest the bone marrow cells from Step 6.4 and transfer the media containing non-adherent cells into sterile 50-ml tubes.
2. Wash the plates vigorously with 10 ml PBS and collect in the 50 ml conical from step 9.1. If necessary, repeat the wash step.
3. Centrifuge the cells at 300 x g for 10 min at 4 °C, decant the supernatant, resuspend cells in a small volume (1 – 3 ml) complete DMEM 20% (vol/vol) FBS, and count. Anticipate 50 – 75% recovery from day 0. Resuspend bone marrow at 1 x 10⁶ cells per 1 ml of the retroviral supernatant containing cytokines and Hexadimethrine bromide.

10. Bone Marrow Transduction (Day 1)

1. Plate the retroviral supernatant/bone marrow mixture at a volume of 3 ml per well in a six-well tissue culture plate. Wrap the plates in plastic wrap to minimize gas exchange during the centrifugation in step 10.2.
2. Spin the wrapped six-well plates at 1,000 x g for 60 min a 37 °C.
3. After the spin is complete, remove the plastic wrap and place the plates in a 37 °C CO₂ incubator for 24 hr.

11. Retroviral Supernatant and Transduction (Day 2)

1. After 24 hr, collect fresh viral supernatant from the viral producer cell line. Filter the retroviral supernatant using a 10-ml syringe and a 0.45-μm syringe filter. Supplement the filtered retroviral supernatant with IL-3 (20 g ml^-1), IL-6 (50 g ml^-1) and SCF (50 g ml^-1) and Hexadimethrine bromide (6 g ml^-1).
2. Next, carefully remove with pipette or aspirate the top 2 ml of media from each well of the six-well plate containing the bone marrow cells.
   NOTE: Work carefully not to aspirate the bone marrow, which is usually concentrated in the middle of the well. Alternatively, the removed supernatant can be spun down to recover any lost bone marrow cells.
3. To each well in the bone marrow 6-well plate add 3 ml of fresh viral supernatant containing cytokines and Hexadimethrine bromide. Wrap the plates in plastic, and repeat the spin transduction at 1,000 x g for 60 min a 37 °C. Unwrap the plates, and incubate cells at 37 °C and 5% CO₂ overnight (about 24 hr).

12. Addition of Fresh Supplemented DMEM (Day 3)

1. After 24 hr, remove the top 2 ml of media from each well of the six-well plate containing the bone marrow cells, as in step 11.2. Replace the removed media with fresh DMEM 20% (vol/vol) FBS supplemented with final concentrations of IL-3 (20 g ml^-1), IL-6 (50 g ml^-1) and SCF (50 g ml^-1).
2. Incubate bone marrow plates at 37 °C CO₂ incubator for another 24 hr.

13. Harvest Transduced Bone Marrow (Day 4)

1. After 24 hr, collect the bone marrow cells from the six-well plates. Wash the plates vigorously with PBS to remove all non-adherent cells.
   1. Centrifuge the cells at 300 x g for 10 min at 4 °C and decant the supernatant.
   2. Resuspend the bone marrow in a small volume (1 ml) of PBS + 0.5% (vol/vol) heat-inactivated FBS. Keep cells on ice.
2. Take a 10-μl sample of each bone marrow condition and count the cells using a Neubauer counting chamber. Assuming 1 – 2 donor per recipient, the yield will be sufficient to transfer at least 4 x 10⁶ cells per recipient mouse. Resuspend the bone marrow in a sufficient volume of PBS + 0.5% (vol/vol) heat-inactivated FBS to inject 200 – 300 μl per mouse.
   1. Inject at least 2 x 10⁶ cells with a transduction efficiency of 25% (can inject up to 8 x10⁶ cells) per mouse intravenously.
      NOTE: We routinely inject five recipient mice per two 6-well plates of bone marrow cells. If the yield is substantially lower, more donor mice should be used until the sufficient bone marrow number can be obtained. In order to achieve optimal reconstitution, at least 5 x 10⁵ transduced (fluorescent positive) should be injected into each recipient.
3. With a micro pipettor, take a 10-μl sample of each bone marrow condition and analyze transduction efficiency by flow cytometry based on expression of the fluorescent marker (Figure 1A)¹¹.
   NOTE: Generally, the percentage of fluorescent positive cells is between 25% and 70%, depending on the construct and retroviral titer. The number of transduced bone marrow cells necessary to reconstitute a sub-lethally irradiate mouse can vary depending on the transduction efficiency. The lower the transduction efficiency, the more bone marrow cells required and conversely, the higher the bone marrow transduction efficiency, the lower number of cells required for reconstitution. Transduction efficiency below 25% is suboptimal and may result in insufficient reconstitution and survival. In order to ensure optimal bone marrow recovery and transduction efficiency, use freshly prepared tissue culture media and cytokines. When expressing a new TCR utilizing the retrogenic system, the selection of that TCR in vivo is unknown. Depending on each individual TCR affinity, TCR selection in the thymus and peripheral T cell expression will vary¹⁷.

14. Mouse Irradiation, Bone Marrow Injection and Care

1. Irradiate mice the day before bone marrow injection (Same day as Step 13). Use the following doses: C57BL/6 mice (and other wild type strains), 900 rads; Rag1^-/- mice, 500 rads; scid mice, 300 rads).
   1. Place recipient mice on Amoxicillin (50 mg/kg/day) or another antibiotic water treatment before irradiation, and keep on antibiotics for the first two weeks after bone marrow injection.
      NOTE: Optimal irradiation dose may have to be determined empirically.
2. For injection, load the cells from Step 13.1 into a 1-ml syringe using blunt needles immediately before injection, then change the needle to 27-gauge for injection, expel any air to avoid air embolism. Heat mice under a heating lamp and inject 0.2 ml of bone marrow per mouse intravenously.
   NOTE: Do not let cells sit in the syringe for any length of time or the cells will settle.
3. Monitor the mice weekly to ensure that they remain healthy.
4. After 5 – 6 weeks, bleed the mice to check for reconstitution on the basis of GFP+/ametrine+ and TCR co-expression (Figure 1B)¹¹.