Cells and mice.
Bone marrow cells were flushed from the femurs and tibias of 5–8-wk-old mice and were grown in RPMI 1640 medium (Sigma-Aldrich) supplemented with 20% heat-inactivated FCS and 50 μM 2-mercaptoethanol (Sigma-Aldrich).
AID-deficient BALB/c Byj mice were obtained from M. Nussenzweig (The Rockefeller University). BALB/c Byj congenic controls and NF-κB–deficient (p50−/−) C57BL/6 mice and IFN-γ–deficient C57BL/6 mice with appropriate congenic controls were obtained from The Jackson Laboratories. IFN-αR–deficient animals were a gift from David Levy (New York University Medical School, New York, NY), and MyD88-deficient mice and MyD88−/−Trif−/− mice along with congenic controls were a gift from S. Akira (via M. Nussenzweig). All animal research has been approved by the Rockefeller University Institutional Animal Care and Use Committee and was in compliance with the relevant federal guidelines.
Virus production and infections.
Replication-deficient Ab-MLV was generated by transfection of 293T cells with pMIG–Ab-MLV retroviral construct (a gift from Naomi Rosenberg, Tufts University School of Medicine, Boston, MA) and pCL packaging DNA (1:1 ratio) using Lipofectamine according to the manufacturer's protocol. Cells were cultured for 48 h, and the supernatant was filtered through a 45-μm filter and used as viral stock for infection of target cells.
Viral titers (infectious U/ml) were determined by counting the number of gfp+ cells that arose after infection of 3T3 cells with different dilutions of viral stock (multiplied by the dilution factor). For Ab-MLV infections, 2 × 106 bone marrow cells were challenged for 2 h with a multiplicity of infection of 0.1.
Agarose transformation assay of bone marrow cells.
2 × 106
freshly prepared bone marrow cells from wild-type and congenic MyD88−/−
-deficient mice were infected with equivalent titers of Ab-MLV for 4 h and seeded in 0.3% agar (United States Biological) containing RPMI–20% FCS–50 μM β-mercaptoethanol as described previously (28
). Macroscopic colonies of transformed pre–B cells were counted between 9 and 12 d postinfection.
Bone marrow cells were isolated from 5–6-wk-old wild-type and congenic MyD88−/− mice and were infected for 2 h with equivalent titers of replication-deficient Ab-MLV. After infection, the cells were washed once with PBS and were injected intravenously into lethally irradiated (800 rads) C57BL/6 8–10-wk-old female mice. Five recipients from each strain were injected with mock-infected bone marrow cells, and 20 recipients from each strain were injected with virus-infected bone marrow cells (1 million bone marrow cells/mouse). Mice were monitored daily and analyzed soon after death by gross pathology, histology, and FACS.
Flow cytometry analysis.
For the quantification of gfp+ B cells, we stained bone marrow cultures with anti–mouse B220 PE-conjugated antibody (BD Biosciences). 7-amino actinomycin D was used for dead cell exclusion. FACS analysis was done on a FACSCalibur (Becton Dickinson) using Cellquest software (Becton Dickinson).
Quantitative (real-time) PCR.
Bone marrow cells from 3–5-wk-old IFN-γ−/−, IFN-αR−/−, MyD88−/−, or MyD88−/−Trif−/− mice were infected with Ab-MLV as described in earlier paragraphs. The cells were collected at different days postinfection and stained with anti–mouse B220 PE-conjugated antibody. Cell sorting was performed on a FACSVantage (Becton Dickinson).
Total RNA was extracted from gfp+
samples using Trizol (Invitrogen), according to the manufacturer's instructions. RNA was treated with DNase I (Promega) at 1 U/μg. Reverse transcription was performed using Superscript III reverse transcriptase (Invitrogen) and random hexamers. Primers for AID amplification were as in reference 20
. Results were normalized to GAPDH.
Phosphorothioate-stabilized CpG oligos (TCCATGACGTTCCTGATGCT) and phosphorothioate-stabilized non-CpG oligos (GCTTGATGACTCAGCCGGAA) were purchased from MWG Biotech. Interferon α-A/D was purchased from Sigma-Aldrich. Double stranded poly(I):poly(C) was purchased from GE Healthcare.
NF-κB chromatin immunoprecipitation (ChIP).
Chromatin immunoprecipitation (ChIP) assays were preformed using the ChIP kit from Upstate Biotech in conjunction with antibodies to NF-κB–p50 (also from Upstate Biotechnology) or with an isotype control antibody. The assay was done according to the manufacturer's instructions.
The following oligonucleotide pairs were used: 5–NF-κB, 5′-CCCCTCCACTGCCAAGCACAGC and 3–NF-κB, 5′- GCCCCCATCCTCCTTCTTCCTC to amplify a 354-bp region of the AID promoter surrounding the NF-κB binding site and 5-APO, 5′-GCCCAATGTGGGTGGTGCCAC-3′ and 3-APO, 5′-CTCAGATTTGAGATCATTCTCTCCAAG to amplify a 313-bp region of the APOBEC1 promoter, which includes an APOBEC1-specific NF-κB binding site. PCR amplification reactions within the linear range were loaded onto agarose gels that were subsequently stained with SyBR green dye (Molecular Probes) and visualized using a Typhoon Fluor Imager.
NF-κB luciferase reporter assays.
For NF-κB luciferase reporter assays, we constructed replication-deficient retroviral constructs carrying the firefly luciferase gene downstream the AID promoter region (nucleotides −1500 to −1) with the κB binding site intact (, wt κB Luc) or mutated (, mut κB Luc). These constructs were transfected into 293T cells to produce two varieties of murine stem cell virus–red virus, which were used to infect bone marrow cells in the presence or absence of Ab-MLV.
7 d after infection, cells were harvested and resuspended in PBS buffer with a proteinase inhibitor mixture (Sigma-Aldrich). Luciferase activity was determined by using the Promega luciferase assay kit on cell lysates (which were prepared as per the manufacturer's instructions). Luminescence was read on a Wallac 1450 Microbeta Trilux luminometer. Protein concentration in lysates was determined by Bradford protein assay (Bio- Rad Laboratories).