Fig. 1
From: CD30 influences germinal center B-cell dynamics and the expansion of IgG1-switched B cells
Generation of a mouse strain expressing CD30 on the cell surface of B cells. A Schematic representation of the generation of CD30//CD19-Cre mice. cDNA for CD30 was introduced into the Rosa26 locus via homologous recombination in embryonic stem (ES) cells. Upstream of the CD30 transgene, a loxP-flanked transcriptional/translational stop cassette was inserted. Downstream of the CD30 transgene, an IRES element along with a truncated form of the human hCD2 gene was inserted to enable tracing of the cells in which the stop cassette was deleted. Following removal of the stop cassette, the transgenes are transcriptionally controlled by the CAG promoter. Transgenic ES cells were utilized to generate CD30stopfl/+ mice. The expression of CD30 in all B cells was achieved by mating CD30stopfl mice with CD19-Cre mice, resulting in CD30stopfl/+//CD19-Cre+/‒ (CD30//CD19-Cre) mice. B Histogram overlay of hCD2 expression in CD30//CD19-Cre mice (red) compared with that in CD19-Cre control mice (blue), demonstrating that ~85% of all splenic B cells have the stop cassette deleted. A representative of 18 flow cytometric analyses is shown. C Protein extracts of splenic B cells were separated via SDS‒PAGE, subjected to Western blotting and detected with an anti-CD30 antibody. The transgenic CD30 protein was detected at the expected size of 110 kDa. The smaller band at ~70 kDa is likely the unglycosylated form of CD30. Tubulin was used as a loading control. D Histogram overlays of hCD2 expression in CD19+ (green or red, respectively) and CD19− (gray) splenic cells in CD30stopfl+/− (CD30+/−) mice and CD30//CD19-Cre mice. Since the hCD2 protein is translated from the same RNA as CD30 it is very likely, that the transgenic CD30 is not expressed in the absence of Cre. E To detect transgenic CD30 expression on the surface of different B-cell populations, splenic B cells were separated into three subpopulations. FoB cells (CD23+CD21+), MZB cells (CD23−CD21high) and CD23−CD21− cells (transitional B cells, B1 cells, and plasmablasts) are gated as illustrated in the FACS plot on the left side. The pregating of the cells is shown in Supplementary Fig. 3a. The histogram overlays illustrate CD30 expression in the indicated populations from CD30//CD19-Cre (red) and CD19-Cre control mice (blue). A representative of 16 flow cytometric analyses is shown
