Figure 1.
Full-length recombinant HEL-IgM-BCR expression cassette.
(A) The HEL-specific Ig chains are linked by the Furin–FMDV-2A self-cleaving sequences. Both Ig chains have a signal peptide sequence, but only the downstream Igκ chain possesses a stop codon. (B) During translation, the first cleavage event occurs at the FMDV-2A peptide sequence; the 2A peptide remains attached as remnant to the C-terminus of the upstream Igμ chain. (C) A second cleavage event, initiated at the Furin site, finally yields both Ig chains without further attachments that can be assembled to build the HEL-IgM-BCR.
Figure 2.
In vitro surface expression of the recombinant HEL-IgM-BCR.
The pro-B cell line R5B, deficient for endogenous BCR, was infected with virus-containing supernatant encoding for the recombinant HEL-specific IgM-BCR. 24 h post-infection, cells were either analysed by flow-cytometry or western blot analysis. The WEHI-231 B-cell lymphoma served as positive control. Staining for Igμ and Igκ showed surface (A) as well as intracellular expression (B) of the recombinant BCR. Numbers in quadrants indicate the percentage of cells expressing the respective marker. (C) Lysates of infected R5B cells were analysed for κ- and μH-chain expression. Expression of the Ig chains was only detected in lysates of cells infected with the recombinant HEL-IgM-BCR. Incubation with an anti-GFP antibody was used as internal control. Results are representative of three independent experiments.
Figure 3.
Analysis of HEL-IgM retrogenic mice showed only weak expression of the retrogene.
Retrogenic mice were generated as described in the Materials and Methods section. Analysis was performed 6- to 8-weeks post-reconstitution. Single cell suspensions of spleen and lymph nodes were stained with fluorochrome-conjugated antibodies and analysed by flow cytometry. Transgenic HEL-IgM-BCR as well as wildtype C57BL/6 mice served as controls. Results for splenocytes are displayed. (A) Representative surface staining for CD19 and Igμb. Transduced cells are GFP+. Numbers in quadrants indicate the percentage of cells expressing the respective marker. Dead cells were excluded from the analysis. (B) Intracellular staining for the same parameters as in (A). Cells are gated on CD19+ cells. (C) Surface staining for surface markers other than B cells (CD3, CD4, CD8, CD11c, Gr-1). (D) Rag2-/- mice served as donors and recipients instead of C57BL/6 wt mice. Flow cytometric analysis for same parameters as in (A) as well as for Igβ. Results shown are representative of two independent experiments.