Engineered B Cell Lines for Antibody Research

DT40^Cre1, which displays increased Ig gene conversion due to a v-myb transgene and contains a tamoxifen-inducible Cre recombinase, has been described previously (Arakawa et al. 2001 (link)). DT40^Cre1AID^–/– was generated by the targeted disruption of both AID alleles of DT40^Cre1 (Arakawa et al. 2002 (link)). AID^R was derived from DT40^Cre1AID^–/– after stable integration of a floxed AID-IRES-GFP bicistronic cassette, in which both AID and GFP are expressed from the same β-actin promoter. AID^RψV^– was derived from AID^R by transfection of pψVDel1-25 (see Figure 1A). Stable transfectants that had integrated the construct into the rearranged light chain locus were then identified by locus-specific PCR. Targeted integration of pψVDel1-25 results in the deletion of the entire ψV gene loci starting 0.4 kb upstream of ψV25 and ending 1 bp downstream of ψV1. AID^RψV^partial was produced in a similar way as was AID^RψV^–, by transfection of pψVDel3-25, which, upon targeted integration, leads to a partial deletion of the ψV loci starting 0.4 kb upstream of ψV25 and ending 1 bp downstream of ψV3. Cell culture and electroporation were performed as previously described (Arakawa et al. 2002 (link)). XRCC3^–/– was derived from DT40^Cre1 by deleting amino acids 72–170 of the XRCC3 gene following transfection of XRCC3 knockout constructs. Clones that underwent targeted integration were initially identified by long-range PCR, and the XRCC3 deletion was then confirmed by Southern blot analysis.