pRetroX-IRES-ZsGreen1

Both non-immune "natural" and antigen-induced "immune" IgM are important in preventing infection and modulating immune responses to self-antigens. The role of its Fc receptor (FcµR) in these IgM effector functions has been explored. In this study, by exploiting differences in IgM ligand binding of human (constitutively bound) and mouse (transiently bound) FcμR, the researchers replaced non-conserved amino acid residues of human FcµR with mouse equivalents before the establishment of cell lines stably expressing mutant or wild-type (WT) receptors. The resulting cell surface expression and IgM binding of eight different mutant FcμR-bearing cells were compared with WT receptor-bearing cells, as assessed by flow cytometry, using receptor-specific mAbs and IgM paraproteins as ligands.

In this study, the coding sequence of human FcµR (huFcµR) and mouse FcµR (moFcµR) cDNAs was flanked by the restriction enzyme sites of BglII (for huFcµR) or SacII (for moFcµR) and of ClaI (for both) at the 5' and 3' sites, respectively, and was synthesized as a wild type (WT). HuFcμR cDNAs encoding the following substitution mutations with the corresponding moFcμR aa residues: KVEG24-27QLNV, E41Q, M42L, EM41-42QL, N66-, KQYPR79-83TPCLD, Y81C and N109K were similarly synthesized. The BglII/ClaI cut, WT and mutant huFcμR cDNA inserts and the SacII/ClaI cut WT moFcμR cDNA insert were then subcloned into the pRetroX-IRES-ZsGreen1 retroviral expression vector and transfected into the PLAT-E packaging cell line, before transduction in the BW5147 mouse thymoma line. Only the pRetroX-IRES-ZsGreen1 vector (no inserted cDNA) was also transduced as an empty vector control. BW5147 cells stably expressing GFP were enriched by fluorescence-activated cell sorting (FACS). Seven N-terminal FcμR mutants were then quantitatively assessed for GFP and FcμR expression and IgM binding activity.

Figure 1. Quantitative evaluation of GFP and FcμR expression and IgM binding activity of seven N-terminal FcμR mutants. (Skopnik C M, et al. 2021)