Extrachromosomal Eukaryotic DNA Substrates for Switch Recombination: Analysis of Isotype and Cell Specificity

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Abstract
Switch recombination in B lymphocytes is a complex process directed by signals provided by cytokines and/or TH cells. To analyze these signals in an in vitro system, we have developed extrachromosomal eukaryotic substrates for switch recombination that replicate autonomously in murine cells and present pairs of switch (S) regions in an accessible mode. Switch recombination within the S regions results in the expression of the selectable neo gene. The results presented here indicate that substrates containing either Sμ and Sγ2b, Sμ and Sγ2a, or Sμ and Sα undergo switch recombination with similar frequencies in the pre-B-cell line 18-81, which has been previously reported to specifically switch to IgG2b. This indicates that, rather than expressing a γ2b isotype-specific recombinase, the 18-81 cells express a switch recombinase capable of acting on any accessible S region, supporting the accessibility model. The extrachromosomal substrates were rearranged in the 18-81 cells, but not in murine myeloma, T-cell, or fibroblast cell lines, supporting the idea that switch recombination is indeed regulated in a cell- and developmentally specific manner. Restriction enzyme analysis of the plasmid DNA recovered from the selected cell lines suggested multiple recombinational events, with most patterns in agreement with deletions within one or both switch regions.