Figure 3. Untagged ΔAID has DN activity, induces very few Sµ DSBs, and does not cause degradation of endogenous AID.

(A) Compilation of IgG1 and IgG3 CSR results (+SEM) for the untagged DAID in WT cells relative to CSR in cells expressing pMIG. The control retrovirus pMIG does not affect CSR significantly, as GFP-negative cells switched similarly to cells transduced with pMIG (not depicted). In these experiments, cells were activated to switch for 24 hrs, transduced and harvested 2 days later. The procedure for gating is illustrated in Fig 7A. In addition, we gated on the brightest GFP+ cells (~50% of the cells). Results from two independent experiments (2 mice) with two cultures each are shown. (B) LM-PCR assay of Sµ DSBs in aid^−/− cells induced to switch to IgG3 transduced with untagged AID, ΔAID or the empty retrovirus pMIG. Transduction was performed on day 2 after activation, and cells harvested on day 3. The line indicates where the image was cut to remove irrelevant lanes. Three fold titrations of input DNA were performed, and the mb-1 gene was amplified as an internal control for template input. The mb-1 PCR bands shown were obtained by electrophoretic analysis on a QIAxcel Advanced instrument, which subjects each sample to electrophoresis in a capillary, and provides an image and quantitation of each lane. (C) LM-PCR assay of Sµ DSBs in aid^−/− cells induced to switch to IgG3 transduced with AID-ER, ΔAID-ER or the control retrovirus ER. Methods similar to B. (D) Westerns blot of 80 µg of total cell extracts of aid^−/− and aid^+/+ cells transduced with untagged AID and ΔAIDexpressed in the retrovirus pMIG, and then probed with antibody to AID and to Grb2 for loading controls. Cells were cultured and transduced as in A, but similar results were observed if cells were cultured for 2 days prior to transduction, and harvested one day later.