Fig 1: CRISPR-Engineered Hyperstabilized cyclin D3 Mutant Mice Increase DZ Expansion(A) Generation of Ccnd3T283A/+ mice was achieved by microinjection of Cas9 mRNA, Ccnd3 sgRNA, and Ccnd3T283A ssODN into zygotes from superovulated C57BL/6 female mice.(B) DNA sequencing chromatogram shows the point mutation and resulting amino acid change in a heterozygous animal for Ccnd3T283A/+.(C) Assessment of cyclin D3 protein levels in Ccnd3T283A/+ mice. The B splenocytes from Ccnd3T283A/+ or Ccnd3+/+ mice were cultured in the presence of indicated stimulants for 16 h, and cell lysates were probed for cyclin D3 (and ß-actin as a loading control). Data are representative of two independent experiments.(D) Irradiated (10 Gy) C57BL/6 recipient mice were reconstituted with bone marrow from Ccnd3+/+ or Ccnd3T283A/+ mice. After 6 weeks, mice were immunized with SRBCs and analyzed 10 days later. Single cells were gated on B splenocytes (B220+) to determine GC B cell frequency based on GL7+FAS+ expression.(E) Quantification of (D) GC B cells in Ccnd3T283A/+ mice compared with Ccnd3+/+ control mice (n = 8).(F) Zonal distribution of GC B cells (shown in D) based on relative expression of CXCR4hiCD86lo (DZ) and CXCR4loCD86hi (LZ).(G) Quantification of (F) GC B cells respective DZ/LZ ratio (n = 8).(H) Antibody titers of SRBC-specific IgM or IgG1 in sera from Ccnd3+/+ or Ccnd3T283A/+ mice (n = 8).(I) Immunofluorescence of GC reaction in Ccnd3T283A/+ and Ccnd3+/+ mice. Mice were immunized with NP-CGG and analyzed on day 14. Splenic follicles with B cells (B220+) and GC B cells (PNA+) are shown. CD35 expression on follicular dendritic cells depicts the LZ of the GC. Scale bar, 100 µm. Data are representative of three independent experiments.