Generation of T Cell Receptor Retrogenic Mice: Improved Retroviral Mediated Stem Cell Gene Transfer

Abstract

The utilization of retrogenic mice offers a rapid and flexible approach to T-cell receptor transgenic mice. By transducing bone marrow progenitor cells with retrovirus that encodes a given TCR alpha and beta subunit, TCR retrogenic mice can be generated in as little as 4-6 weeks whereas conventional TCR transgenics can take 6 months or longer. In this updated protocol, we have tried to increase the efficiency of the bone marrow transduction, and bone marrow reconstitution. The main departure from the previous protocol is implementation of the spin transduction with the viral supernatant instead of co-culture with the viral producer cell line. The changes in this protocol improve bone marrow viability, increase consistency of the bone marrow transduction and bone marrow engraftment, as well as, reduce the ratio of bone marrow donor mice per bone marrow recipients.

Introduction

Over the past six years a number of publications have emerged that utilized retrogenic mice, reviewed in ref. 1. In an effort to maximize the transduction efficiency and the overall reconstitution of retrogenic mice, we have adjusted and modified portions of the original protocol published by Holst et al ². To this end, an updated protocol for the Retroviral Mediated Stem Cell Gene Transfer section is detailed in this Protocol Update article. The updated protocol has been successfully utilized for over three years in multiple published (refs 3 and 4) and unpublished projects. Specifically, steps 36-45 of the original protocol were modified. In summary, the revised protocol utilizes only the legs, hips and the upper arms as sources of bone marrow. These particular bones yield sufficient numbers of bone marrow cells for at least a 1:1 donor to recipient transfer and in some instances as few as 1:2 depending on the mouse genetic background. In addition, although the retroviral producer cell co-culture system was sufficient for bone marrow transduction and host reconstitution, we were not content with the overall transduction efficiency and bone marrow viability and thus modified this portion of the protocol. We now suggest using filtered retroviral supernatant supplemented with IL6, SCF, and IL-3 and polybrene to spin-transduce the bone marrow for two consecutive days in order to achieve approximately a 2-5 fold increase in transduction efficiency.

Materials

The materials used in the original protocol are required plus the following additions.

Reagents

• 6 well Cell Culture Cluster Flat Bottom with Lid (Costar, cat. no. 3516)

• All Purpose Laboratory Wrap (Fisherbrand, cat. no. 01810)

• Hanks’ Balanced Salt Solution with calcium and magnesium (Corning/Cellgro, cat. no. 21-020-CV)

Procedure

Retroviral Mediated Stem Cell Gene Transfer

1. Plate out viral producer cell lines on 150 mm plates. Allow enough time to passage producer cell lines to obtain 2 confluent plates (3× 10⁶ producers per plate) per 5 recipient mice, the same day as harvesting bone marrow. All steps throughout the protocol are assuming 5 recipient mice per group.

CRITICAL STEP: Ensure that producer cells are growing well, i.e. in an exponential growth phase, before injecting 5-fluorouracil (5-FU) into the mice, if the producer cell lines are not healthy or overgrown, transduction of bone marrow will not occur efficiently.

• 2Weigh the donor mice (5-12 weeks old), to determine the amount of 5-FU required.
• 3Make a 10 mg/ml working solution of 5-FU in sterile PBS, with each mouse receiving 0.15 mg per gram of body weight. Inject appropriate volume of 5-FU intra peritoneal (IP) using a 27 gauge needle 72hr prior to harvest of bone marrow. Injection 96hr prior to harvest of bone marrow resulted in slightly lower bone marrow cell number recovery, but similar transduction efficiency and reconstitution. Injection 48hr prior to harvest of bone marrow resulted in higher cell number recovery, but similar cell number recovery by the end of transduction protocol, and suggested incomplete apoptosis of rapidly dividing cells of the hematopoietic lineage in the bone marrow prior to bone marrow isolation.

Standard Bone Marrow Extraction Procedure

• 4Cut out the femur and tibia and remove all surrounding tissue using scissors, forcepts, and paper towel to obtain clean bones. Keep hydrated in HBSS + 5% FBS. CAUTION All further steps should be carried out in a biological safety cabinet to prevent contamination of bone marrow.
• 5Cut both ends off each bone, and flush bones by inserting a 10 ml syringe containing HBSS + 5% FBS with a 25 gauge needle into the end of the bone. Flush into a 70μm strainer resting in a 50 ml tube, until the red plug of bone marrow is removed. Repeat until all bone marrow is released from the bones. Using a plunger from a 1ml syringe, work the cells through the 70μm strainer. Rinse the Strainer with 10 mls of HBSS + 5% FBS to ensure all bone marrow cells are filtered into the 50 ml tube.

CRITICAL STEP: Processing of bone marrow and all subsequent steps should be kept as sterile as possible.

• 6Centrifuge cells at 300g for 10 minutes at 4°C, decant supernatant and resuspend in Gey's solution (or comparable Red Cell Lysis buffer). After 3 minutes incubation at room temperature (RT), quench the Gey's solution with 10 ml of 20% C–DMEM. Spin the cells at 300 g for 10 minutes at 4°C, aspirate and resuspend in 20% C–DMEM to count cells using a Neubauer counting chamber. General yield should be approximately 2-3 × 10⁷ per B6.Rag-/- mouse, however yield can vary among different strains (for instance, we recover approximately half as many cells from NOD.scid mice). Removing red blood cells improves transduction efficiency.
• 7Plate out bone marrow cells at 4 × 10⁷ cells per 150 mm plate, in 30 ml of 20% C–DMEM, supplemented with IL-3 (20 ng/ml), IL-6 (50 ng/ml) and SCF (50 ng/ml) for 24 hr in a 37°C incubator.
• 8Plate out 3 × 10⁶ producer cells per plate, with two plates per 5 recipient mice. Plate cells in 18 ml 20% C–DMEM for 24 hr in a 37°C incubator.
• 9After 24 hours, filter the retroviral supernatant using a 10ml syringe and a 0.45 μm filter. Replace the removed retroviral supernatant with 18 ml fresh 20% C– DMEM. Supplement the filtered retroviral supernatant with IL-3 (20 ng/ml), IL-6 (50 ng/ml), SCF (50 ng/ml) and polybrene (6 μg/ml).

Critical step: Always use freshly thawed cytokines. This can be achieved by first reconstituting, then immediately aliquoting and freezing new cytokines to be thawed for later use. We do not suggest using cytokines that have been freeze thawed more than once or kept at 4°C for more than two weeks.

• 10After the retroviral supernatant is prepared as in step 9, harvest bone marrow cells from the Step 7 plates, by decanting media into sterile 50 ml tubes, then washing and scraping plates using a cell scraper with 10 ml 20% C–DMEM. Ensure the entire surface of the plate has been scraped, decant 20% C–DMEM into same 50 ml tubes, and wash the plates with 5 ml 20% C–DMEM to recover remaining cells. Centrifuge cells at 300 g for 10 minutes at 4°C, decant supernatant and resuspend to approximately 4 × 10⁷ cells/ml in 20% C–DMEM. Add 0.75-1.0 × 10⁶ bone marrow cells per 1 ml of the retroviral supernatant containing cytokines and polybrene.
• 11Plate the retroviral supernatant/bone marrow mixture at 3mls per well of a 6 well tissue culture plate. Wrap the plates in plastic wrap to minimize gas exchange.
• 12Spin the wrapped 6 well plates at 1000 g (2500 RPM) for 90 minutes at 37°C, then immediately place the plates in 37°C incubator for 24hrs.
• 13After 24 hours, repeat step 9. Filter the retroviral supernatant using a 10ml syringe and a 0.45 μm filter. Supplement the filtered retroviral supernatant with IL-3 (20 ng/ml), IL-6 (50 ng/ml), SCF (50 ng/ml) and polybrene (6 μg/ml).

Optional: Replate the viral producer cells line in fresh 20% C–DMEM to use for a third spin transduction after 24 hours. At this point viral producer cell lines will be confluent and will have to be split again into 3 × 10⁶ cells per 150 mm plate in 18 ml 20% C–DMEM.

• 14Next, carefully remove and collect the top 2 ml of supernatant from each well of the 6 well plate containing the bone marrow cells. Spin this supernatant down to collect any cells. Resuspend the cells with the freshly filtered retroviral supernatant from step 13, and transfer back to the 6 well plates containing the remaining bone marrow.
• 15Repeat step 12.

Optional: Repeat steps 13-15 for a third day of spin transduction. This third spin may not be necessary as we have observed minimal increase in transduction efficiency after the third spin.)

• 16After 24 hours carefully remove and collect the top 2 ml of supernatant from each well of the 6 well plate containing the bone marrow cells. Spin this supernatant down to collect any cells. Decant supernatant and resuspend the cells with fresh 20% C–DMEM supplemented with final concentrations of IL-3 (20 ng/ml), IL-6 (50 ng/ml), SCF (50 ng/ml). Transfer the media containing cytokines and bone marrow back to the 6 well plates. Place 6 well plates directly into 37°C incubator for 24hrs.
• 17After 24 hours, collect the bone marrow cells from the 6 well plates and centrifuge cells at 300 g for 10 minutes at 4°C and decant supernatant. For reconstitution of 5 mice, resuspend bone marrow in 1 ml PBS + 0.5% FBS supplemented with heparin (20 units/ml) per group, which will allow injection of 0.2 ml per mouse.
• 18Take a 10 μl sample, and count cells using a Neubauer counting chamber. Yield should be enough to transfer at least 4 × 10⁶ cells per recipient mouse. We routinely inject 5 mice per 2 plates of bone marrow cells. If the yield is significantly lower, more donor mice should be used until this number can be obtained. Take another 10 μl sample to analyze by flow cytometry for GFP⁺ cells. Generally the percentage of GFP⁺ cells is between 25-70% depending on the construct and retroviral titer. Lower numbers of bone marrow cells may be sufficient to reconstitute mice depending on the overall transduction efficiency of the progenitor population. Transduction efficiency below 25% may hamper successful reconstitution.
• 19Irradiate mice the day before injection (We use the following: Rag-1^−/− - 500 rads; C57BL/6 - 900 rads; NOD-scid – 300 rads).

Critical step: If mice are not in an SPF/helicobacter-free facility, they should be placed on Sulfatrim water prior to irradiation and injection, and for the duration of the experiment. Optimal irradiation dose may have to be determined empirically.

• 20For injection, load cells from Step 18 into 1 ml syringes using blunt needles immediately before injection. Inject recipient mice using 27 gauge needles. Heat mice under a heating lamp and inject with 0.2 ml of bone marrow per mouse intravenously.
• 21Monitor mice weekly to ensure they remain healthy after the irradiation.
• 22After 5-6 weeks, bleed the mice to check for reconstitution based on GFP⁺ and TCR co-expression.

Troubleshooting

Ensuring the health of the bone marrow progenitors is critical to the success of the protocol. To this end, ensure cytokines and media are fresh. Cytokines should not be used if older than 2 weeks when kept at 4°C and DMEM should not be more than 4 weeks after supplemented with FCS. It is also critical to spin transduce the plates containing bone marrow cells at 37°C and have them wrapped in plastic wrap to prevent gas exchange as much as possible. Viral producer cell lines should be in the early passage, and not grown to confluency as this will reduce the viral titer.

Anticipated Results

This change to the procedure does not change the overall results of the protocol already extensively discussed in ref 2.