AIDCre-mediated deletion of the miR-15a/16-1 cluster in murine lymphoid cells. (A) Alignment of human and mouse miR-15a-5p and miR-16-5p sequences. (B) Schematic of the strategy used to generate mice with miR-15a/16-1 deletion during B-cell activation. (C) miR-15a/16-1 cluster deletion assessed by PCR in indicated FACS-sorted lymphocyte subpopulations from WT (n = 4) and KO (n = 4) mice. WT locus, 558 bp (bottom band); loxP flanked miR-15a/16-1 cluster, 650 bp (middle band); deleted miR-15a/16-1 cluster, 850 bp (top band). (D) miR-15a, miR-15b, and miR-16 expression in FACS-sorted lymphocyte subpopulations from WT and KO mice, determined by RT-qPCR relative to snoRNA234. Error bars represent standard deviations of 3 independent replicates in a representative experiment. P values were calculated by using the unpaired Student t test. (E) Cre (IHC, brown; counterstain, blue) and miR-15a, miR-15b, and miR-16 expression (ISH, dark purple) in spleen sections of 12-week-old WT and KO mice. One representative example of secondary follicle for each genotype is shown. GCs are highlighted by dotted lines; scale bar, 100 μm. ISH sections were not counterstained to facilitate interpretation (see also supplemental Figure 3A-B). (F) IHC (brown; counterstain, blue) analysis of BCL2 and Ki-67 expression in splenic GCs from mice with indicated genotypes. The BCL2 staining of spleen section from a WT mouse is shown at lower magnification to depict the general pattern of BCL2 expression in murine lymphoid tissue. Note high BCL2 abundance in T cells surrounding periarteriolar sheaths (PS), MZ B cells, and Fo B cells in white pulp (WP) areas, as well as considerably lower BCL2 expression within GCs and red pulp (RP) areas. Scale bar, 50 μm. (G) BCL2 expression in indicated lymphocyte subpopulations from WT and KO mice immunized with SRBCs assessed by immunoblotting. Quantitative differences in protein expression level based on densitometric analysis were normalized to actin and are shown below the blots. Apostrophe indicates longer times of film exposure to exhibit relative differences in protein abundance. Unrelated bands were cut from the immunoblots (dotted line).