Retroviral Transduction of BMMCS

This section can be divided into: (1) the construction of retroviral expression vector; (2) the generation of recombinant retrovirus; and (3) the infection of replicating mast cells with recombinant retrovirus (see Note 4).

3.2.1. Construction of Retroviral Vector

A Moloney murine leukemia virus-based vector, pMX-puro (8,9), has been extensively used for transfection of mouse BMMC. As almost all gene-targeted mice have a "neo" gene cassette in their genome, the retroviral vector must have a different drug-resistance gene, such as the puromycin resistance gene or another way of selection, for example, a green fluorescent protein gene in the bicistronic gene expression allele. The expression of the latter can be detected by flow cytometry or fluorescent microscopy. The most popular vector, pMX-puro (Fig. 1), can accommodate a gene or cDNA in the region

Pmx Neo Vector Map

Fig. 1. The most popular vector, pMX-puro (the original figure was kindly provided by Dr. Toshio Kitamura of the University of Tokyo), can accommodate a gene or cDNA in the region between the BamHI (nucleotide 1884) and NotI (nucleotide 3161) sites.

Multi-cloning site

Fig. 1. The most popular vector, pMX-puro (the original figure was kindly provided by Dr. Toshio Kitamura of the University of Tokyo), can accommodate a gene or cDNA in the region between the BamHI (nucleotide 1884) and NotI (nucleotide 3161) sites.

between the Bam HI (nucleotide 1884) and Not I (nucleotide 3161) sites. Standard molecular biological techniques are used to construct recombinant vectors.

3.2.2. Generation of Recombinant Retrovirus

Recombinant retroviral vectors can be transfected into a packaging cell line to generate infectious virus particles. There are several packaging cell lines available for this purpose: for example, BOSC 23 (10), Phoenix (11), and PlatE (12). Our experience indicates that all of these packaging cell lines yield titers of viruses high enough to produce transfected BMMC in a scale of 5 to 20 x 107 cells after puromycin selection. These numbers of transfectants allow for most of biological and some biochemical analyses. In this section, we will describe our standard protocol using BOSC 23 cells.

3.2.3. Infection of Mast Cells and Selection of Transgene-Expressing Cells

Retroviral genomes can integrate into a host genome only when host cells are replicating. Although IL-3 usually is used as a growth factor to generate BMMC, IL-3 alone is not strong enough to induce vigorous cell cycling for efficient retroviral transduction in BMMC. For this purpose we and others feed bone marrow cells in IL-3 and SCF to generate BMMC that are ready for retroviral infection (hereafter abbreviated as sBMMC).

3.2.4. Day-by-Day Procedures Approximately 10 Days Before Transfection

1. Reconstitute a vial of frozen BOSC 23 cells in DMEM/10% FCS/Gln.

2 Days Later

1. Change medium to GPT selection medium. GPT selection medium: Dulbecco's modified Eagle's medium (DMEM), 10% FCS/Gln, and GPT Selection Reagent (the kit contains 500X mycophenolic acid and 100X aminopterin solution; Specialty Media, Phillisburg, NJ).

BOSC 23 Cells Passage

1. After BOSC 23 cells become confluent, passage every 3 or 4 days by three- to fivefold dilution.

2. Aspirate medium very carefully with Pasteur pipet and wash once with 5 mL of PBS.

3. Add 1 mL of trypsin-EDTA and incubate at room temperature for 1-2 min.

4. Add 3 mL of DMEM and pipet well to separate individual cells. It is very important to prevent cells from making cramps.

5. Centrifuge the cells.

6. Suspend in 8 mL of GPT medium per 100-mm dish.

7. Plate BOSC 23 cells homogenously. Pipet BOSC 23 cells well to prevent them from making cramps.

2 Days Before Transfection

1. Remove GPT selection medium from BOSC 23 cells 48 h before transfection and add DMEM/10% FCS/Gln.

1 Day Before Transfection

1. Plate 5.5-6.5 x 106 cells per 100-mm dish or 2 x 106 cells per 60-mm dish. For each transfection, prepare three plates/cell densities of 5.5 x 106, 6 x 106, and 6.5 x 106.

Day 1

1. Make DNA/Lipofectamine mixtures:

a. Mix 10 pg of DNA with 0.75 mL of OPTI-MEM (Gibco) in a Falcon 2059 tube. Add 20 pL of Plus Reagent (Invitrogen) and incubate at room temperature (RT) for 15 min.

b. Mix 30 pL of Lipofectamine reagent (Invitrogen) with 0.75 mL of OPTI-MEM (Lipofectamine cocktail).

c. Transfer Lipofectamine cocktail to the DNA tube, mix, and incubate at room temperature for 15 min.

2. Add 4.5 mL of OPTI-MEM (final total volume: 6 mL/transfection).

3. Pick up BOSC cell plates of the best condition (~60% confluent).

4. Rinse BOSC cells once with 5 mL of OPTI-MEM and overlay the above described DNA-Lipofectamine complex solution (6 mL) onto the rinsed BOSC cells.

5. Incubate at 37°C for 5 h in a CO2 incubator.

Day 2

1. Replace the medium with 9 mL (per 100-mm dish) of fresh complete medium (DMEM/10% FCS/Gln) at 24 h after transfection.

Day 3

1. At 48 h after transfection, collect the supernatant of BOSC cells and centrifuge for 5 min at 350g at 4°C to remove living cells.

2. Infection cocktail (10 mL): Supernatant of BOSC cells (8 mL), D11 (1 mL), FCS (1 mL), 0.15 mg/mL SCF (3.3 pL), and 4 mg/mL polybrene (freshly prepared; 25 pL).

3. Centrifuge 1 x 107 cells/tube sBMMC, remove supernatant, and suspend the cells in the 10 mL of infection cocktail.

4. Plate sBMMC cells into a 100-mm dish. After 8 h, add 10 mL of fresh sBMMC medium with 10 pg/mL polybrene.

5. Incubate 16 h in 5% CO2 incubator.

Day 4

1. At 24 h after infection, wash the cells, suspend in 25 mL of fresh sBMMC medium, and culture them in a 75-cm2 flask (Falcon cat. no. 3111).

Day 5

1. At 48 h after infection, start to select the cells with puromycin. Add 10 mL of puromycin-containing sBMMC medium to 25 mL of cell culture fluid (final puromycin concentration: 1.5 pg/mL).

DAY 8

1. At 3 d after selection, add 15 mL of sBMMC medium.

DAY 15

1. At 10 d after selection, remove 15-20 mL of supernatant and add 15-20 mL of fresh medium, including puromycin.

Day 19

1. After 14-16 d of selection, the cells usually start to grow well, then select living cells using Ficoll as follows.

2. Collect the cells in a 50-mL tube. Spin down at 300g for 5 min.

3. Resuspend the cells in 6 mL of BMMC medium.

4. Overlay the cell suspension in a 15-mL tube containing 6 mL of Ficoll (Roche).

5. Spin at 300g for 15 min.

6. Save the live cells packed between Ficoll and medium and transfer to a 50-mL tube.

7. Wash the cells twice with 30 mL of BMMC medium.

8. Transfer the cells to a 75-cm2 flask. Keep incubate for several more days in the selection medium.

2 Days Before Using the Cells for Experiment

1. Wash the cells twice with BMMC medium and remove puromycin.

2. Suspend the cells in sBMMC medium, and keep incubating until the day the experiment is performed.

The Day of Experiment

1. Wash the cells twice with BMMC medium to remove SCF from medium.

2. Incubate the cells in BMMC medium with or without IgE for 6-8 h (see Note 5).

3. Stimulate cells with antigen as described in Subheading 3.3.2.

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