Primer sequences were designed to fuse AID/APOBEC in-frame with the mKG fragments at either the N or C terminus. AID and APOBECs were designed as depicted in Fig. 1 and Fig. S1. We generated four constructs (A, B, C, and D) encoding the protein of interest (AID/APOBEC) fused to either the N- or C-terminal fragments Cyproterone acetate of mKG protein, mKG.N (168 aa), and mKG.C (51 aa). There was a 24-aa glycine-rich linker sequence between the mKG fragment and the protein of interest to provide greater mobility to the fusion protein. Primer sequences Cyproterone acetate were designed to fuse AID/APOBEC in-frame with the mKG fragments at either the N or C terminus. The coding sequences for AID/APOBEC were mostly cloned into Cyproterone acetate the KpnI and EcoRI sites of the respective mKG vectors (Code No. AM-1100; MBL). For co-IP experiments, AID was tagged at its C terminus with a FLAG or HA epitope. CSR Assay in Primary B Cells. To analyze the CSR efficiency of WT and mutant AID, primary B lymphocytes were isolated from AID-deficient mice as described above, and cultured at 1.0 106 cells per mL in complete RPMI medium containing 25 g/mL LPS and 7.5 ng/mL IL-4, to undergo class switching to IgG1. For the retroviral transduction, the cells were preactivated before infection by culturing in the presence of LPS and IL4 for 48 h. The retroviral supernatants were prepared, and spleen cell infection was performed using standard protocols. The IgG1 expression was examined by flow cytometry by staining the cells with biotinylated anti-IgG1 (Pharmingen) and APC-conjugated streptavidin (eBioscience) on day 3. The IgG1 switch efficiency was calculated from the infected GFP-positive cells in the live gate. Gene KD in CH12F3-2A Cells and DNA Break Assay. To knock down the expression of specific genes of interest, chemically modified Stealth siRNA oligonucleotides (Invitrogen) were introduced into CH12F3-2A cells using the Nucleofector 96-well electroporation system (Lonza) (62, 63). After electroporation, Rabbit Polyclonal to TPD54 the cells were cultured for 24 h and then stimulated by CIT for another 24 h to induce IgA switching. Cells were stained with FITC-conjugated anti-IgM (eBioscience) and PE-conjugated anti-IgA (eBioscience) and subjected to FACS analysis using a FACSCalibur (BD Biosciences). The IgM-to-IgA switching efficiency was examined in the live cell population. For the LM-PCRCbased DSB assay, the cells were stimulated for CSR as described above, and the live cells were embedded in low-melt agarose plugs and processed for linker ligation as described previously (64, 65). The samples were treated with T4 polymerase (Takara) before linker ligation, and the ligated DNA was subjected to GAPDH DNA PCR analysis to adjust the DNA input before LM-PCR. Threefold dilutions of the input DNA were amplified by KOD-FX-Neo polymerase (Toyobo). The PCR products were separated by electrophoresis on 1% agarose gels and validated by Southern blotting using a 5 S probe; the primers and probe sequences were the same as described previously (64). Acknowledgments We thank Jin Highway and Keiko Yurimoto for excellent technical assistance in the mKG-BiFC work. We also thank Dr. Afzal Husain for support during the writing and for critical reading of the manuscript. This research was supported by Grant-in-aid for Specially Promoted Research 17002015 (to T.H.) and Grant-in-Aid for Scientific Research 24590352 (to N.A.B.) from the Ministry of Education, Cyproterone acetate Culture, Sports, Science, and Technology of Japan. S.M. acknowledges support from the Human Frontier Science Program (HFSP) for his postdoctoral fellowship. Footnotes The authors declare no conflict.
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