Volume 7, Issue 2, Pages 331-342 (February 2001) SC35 Plays a Role in T Cell Development and Alternative Splicing of CD45  Huan-You Wang, Xiangdong Xu, Jian-Hua Ding, John R Bermingham, Xiang-Dong Fu  Molecular Cell  Volume 7, Issue 2, Pages 331-342 (February 2001) DOI: 10.1016/S1097-2765(01)00181-2

Figure 1 Targeting the SC35 Gene in ES Cells and Mice (A) The targeting construct and expected patterns of Cre-mediated homologous recombination. Indicated are the first two SC35 coding exons (black rectangles), loxP sites (black triangles), PCR primers (horizontal arrows), and the 5′ and 3′ external probes. B, BstXI; Bf, BfaI; A, AvrII; P, PmlI; S, SacI; Xh, XhoI; K, KpnI; X, XbaI. (B) Identification of positive ES clones by PCR. ES cell pools were screened by using the primer set (TK303 and P1). Positive control: targeting vector DNA. Molecular marker: 1 kb DNA ladder. (C) Homologous recombination at the 5′ end. The clone #43–3 contains the expected 7.2 kb BstXI-KpnI fragment detected with the 5′ external probe. (D) Confirmation of three loxP sites. DNA from Clone 43–3 was digested with BamHI and EcoRI and blotted with a radiolabled loxP sequence. (E) After Cre-mediated recombination, SacI-digested DNA from selected ES clones was subjected to Southern blotting analysis using both the 3′ (lanes 1–3) and 5′ (lanes 4–6) external probes. (F) Southern blotting analysis of DNA extracted from tails of wild-type (+/+), heterozygous (+/SC35flox), and homozygous (SC35flox/SC35flox) Type II mice. Diagnostic bands for Type II deletion are the 8.2 kb SacI fragment from the 3′ end and the 7.2 kb BstXI-KpnI fragment from the 5′ end Molecular Cell 2001 7, 331-342DOI: (10.1016/S1097-2765(01)00181-2)

Figure 2 Profound Effect of SC35 Deletion on Thymocyte Development (A) Conversion of Type II to Type I deletion in the thymus. The presence of the Lck-Cre transgene was indicated at the bottom. (B) Northern blotting analysis of the SC35 mRNA. (C) Morphology of thymi and spleens of littermates. (D) Reduction of thymus weight. The ratio of thymus (T) to total body weight (TBW) in homozygous mice was reduced to 30% of that in wild-type and heterozygous mice, while the ratio of spleen (S) to TBW remained the same. n, number of mice analyzed in each group. Data were expressed as mean (M) ± standard deviation (SD). (E) Reduction of total thymocytes. The number of thymocytes in SC35Δ/SC35Δ mice was reduced to about 10% of the wild-type level. A modest reduction was also detected with heterozygous animals. The total number of splenocytes was unaffected Molecular Cell 2001 7, 331-342DOI: (10.1016/S1097-2765(01)00181-2)

Figure 3 Block of T Cell Maturation in the Thymus (A) Variable levels of gene disruption in homozygous mice. While Cre-mediated deletion of the SC35 gene in heterozygous mice reached near completion, the deletion efficiency varied among homozygous SC35 mice (compare mice from 3 to 6), indicating a selection pressure against SC35-deleted cells. (B) Flow cytometric analysis of thymic T cells using anti-CD4-PE and anti-CD8-FITC. The mice and their genotypes shown on the top correspond to those in (A). The results demonstrated a severe block at the transition from DN to DP and the defect correlated with the degree of gene disruption. Shown in each quadrant is the percentage of DN, DP, and SP T cells. (C) Quantitation of T cell subpopulations illustrates the percentage increase and decrease of DN and DP cells, respectively, as a result of the genetic depletion of SC35. Data were expressed as mean (M) ± standard deviation (SD). n, number of mice analyzed in each group. (D) Accumulation of CD44−CD25+ cells in the thymus from SC35Δ/SC35Δ mice. Surface expression of CD44 and CD25 on DN-gated thymocytes was analyzed by triple staining with anti-CD44-PE, anti-CD25-FITC, and a cocktail of Tri-Color (TC)-conjugated mAbs to CD4 and CD8. (E) Quantitation of CD44 and CD25 subset populations revealed that genetic depletion of SC35 caused a selective accumulation of CD44−CD25+ cells in the thymus. Data were expressed as mean (M) ± standard deviation (SD). n, number of mice analyzed in each group. (F) Light micrographs of thymus thin sections, showing that the thymus from homozygous mice had a marked reduction of cellularity and lost the cortical-medullary junction in comparison with those from wild-type and heterozygous mice. Cx, cortex; M, medulla Molecular Cell 2001 7, 331-342DOI: (10.1016/S1097-2765(01)00181-2)

Figure 4 Elimination of SC35-Deleted Cells along the T Cell Development Pathway Thymocytes and splenocytes from littermates of indicated genotypes were stained with anti-CD4-PE and anti-CD8-FITC and different T cell populations were sorted by flow cytometry. SacI-digested DNA from each subset was analyzed by Southern blotting using the 3′ external probe. Bands for SC35flox and SC35Δ in each lane were quantitated by densitometry, and the deletion efficiency shown at the bottom was calculated as the ratio of SC35Δ over the sum of SC35flox and SC35Δ Molecular Cell 2001 7, 331-342DOI: (10.1016/S1097-2765(01)00181-2)

Figure 5 Deletion of SC35 Affects T Cell Maturation (A) FACS analysis of splenic T cells revealed a significant reduction of mature T cells. (B) Quantitation of splenic mature T cells. The level of both CD4+ and CD8+ SP cells was reduced in homozygous mice. A modest reduction of CD8+ SP was also observed in heterozygous animals. Data were expressed as mean (M) ± standard deviation (SD). n, number of mice analyzed in each group. (C) Light microscopic photographs (250×) of stained spleens. Paled white pulp (WP) shows reduced cellularity. Arrows indicate the arterioles (A) Molecular Cell 2001 7, 331-342DOI: (10.1016/S1097-2765(01)00181-2)

Figure 6 FACS Analysis of Cell Surface Expression of CD3 and TCRβ and CD45 Single thymocyte suspensions from wild-type (row 1), heterozygous (row 2), or homozygous (row 3) mice were stained with anti-CD3 (column 1), TCRβ (column 2), CD45ALL (column 3), and CD45RB (column 4) monoclonal antibodies. The expression of these cell surface molecules is each plotted as log fluorescence intensity versus cell number. The percentage of cells belonging to distinct groups is indicated. The results show a dramatic reduction of cell populations with low-to-intermediate CD3 and TCRβ (columns1 and 2) and a diminished expression of CD45 isoforms (column 4), although the overall level of CD45 on the cell surface (column 3) was little affected. Shadowed lines show the profiles of unstained thymic cells from wild-type mice Molecular Cell 2001 7, 331-342DOI: (10.1016/S1097-2765(01)00181-2)

Figure 7 SC35 Is Involved in CD45 Alternative Splicing (A) Isolation of CD45RB positive and negative cells. Thymic cells from SC35Δ/SC35Δ mice were double-strained with tricolor labeled anti-CD4 and anti-CD8 and PE-labeled anti-CD45RB antibodies. CD4/8-positive cells were sorted into RB-positive and RB-negative populations using wild-type T cell as reference. The presence and absence of the surface CD45RB were confirmed by FACS analysis of sorted cells. (B) PCR-based genotyping of sorted cells. DNA isolated from sorted thymocytes from a SC35Δ/SC35Δ mouse was analyzed by PCR to determine the ratio of SC35 deleted and undeleted cells (lanes 2 and 3). DNA from thymocytes of a wild-type mouse was used as a control (lane 1). (C) RT–PCR analysis of CD45 alternative splicing. Extracted RNAs were analyzed by RT–PCR using the primer set hybridizing to CD45 exon 3 and 9. The PCR reactions were kept at the linear range by using a minimal number of cycles (25 to 30 cycles) and analyzing the products by hybridization using a probe derived from CD45 exon 8 and 9. CD45R0, the CD45 isoform lacking exons 4 to 6; CD45RB, the CD45 isoform containing exon 5 but lacking exons 4 and 6; CD45 (-ex 4–7), the CD45 isoform lacking exons 4 to 7 Molecular Cell 2001 7, 331-342DOI: (10.1016/S1097-2765(01)00181-2)