Antibody class switch recombinase activity is B cell stage specific and functions stochastically in the absence of 'targeted accessibility' control

Chromosomally integrated retroviral switch (S) substrates have been developed to reveal switch recombinase-like activities (SRLA) in pre-B and mature B cell lines. Switch substrate retrovectors (SSR) contain a long-terminal repeat-driven neomycin (Neo) gene for proviral chromosomal maintenance (pre- and post-S recombination) and a CMV promoter-driven, chimeric hygromycin-thymidine kinase (Hytk) gene (flanked by S-mu and S(gamma)2b recombination targets) to select for (ganciclovir) and against (hygromycin B)S region recombination, The retro-substrates' strong, constitutive promoters ensure that variations in cellular switch recombinase activities are independent of S region accessibility control, By initially selecting for proviral integrants in hygromycin followed by shifting into neomycin + ganciclovir to select for S sequence-mediated deletions, switch recombinations can be specifically forestalled in B cell lines whilst most switch-incompetent cells do not survive secondary selection. A qualitative, direct PCR assay reveals that SSR recombinations are stochastic in B cell lines generating a product array akin to natural CH class switching. A semi-quantitative DC-PCR assay detects a significant recombinase activity only in a restricted set of late stage pre-B and mature B cell lines, BCL1B1 mature B cells have the highest level of recombinase activity with 25% or more of proviral integrants accumulating S-mu/S<(gamma>)S2b substrate recombinations within 10-14 cell generations, The SSR recombinase assay can be performed in a transient fashion wherein extensive, B cell-specific recombination can be visualized within only a few cell divisions post proviral integration, We propose that switch recombinase activity becomes activated during B cell ontogeny independent of or prior to the acquisition of C-H locus accessibility and that endogenous S segment targeting to pre-existing recombinase requires a level of accessibility beyond transcriptional activation.