Simultaneous production of IgM-like BCR and secreted IgM using mutant 2A peptides.

<p>(A) Schematic representation of the IgM version of the Molecular Rheostat Immunoglobulin constructs. Shown at the top is the design of the heavy chain constructs, containing a secreted IgM heavy chain linked via a 2A element to the transmembrane region (41 aa inclusive of the C-terminus) of the membrane form of the IgM heavy chain. 2A: location of self-cleaving 2A elements. See (B), (C), and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050438#pone-0050438-t001" target="_blank">Table 1</a> for the specific 2A peptides and their sequences. Shown at the bottom is the light chain construct, furnishing the b12 κ light chain. CMVp: CMV promoter. LTR: long terminal repeat. MH and EEK promoters: internal B cell specific promoters from human IgM heavy and κ light chain loci, respectively. b12 µ heavy chain: IgM heavy chain with variable region corresponding to that of the b12 broadly neutralizing antibody. (B) IgM surface staining of 293T cells co-transfected with the same amount of the IgM Molecular Rheostat constructs (heavy chain to light chain in 1∶1 ratio by mass) together with a third construct expressing human Igα and Igβ. The cells were harvested 48 hours post-transfection. All IgM Molecular Rheostat constructs produced surface IgM. White: Membrane-bound IgM control. Blue: Molecular Rheostat Constructs. Gray: GFP control. (C) IgM ELISA of supernatants of transfected cells. The constructs expressing different 2A mutants produced different amounts of secreted IgM. IgM: membrane-bound IgM control.