Journal: PLoS ONE

Article Title: Role of the Transcriptional Corepressor Bcor in Embryonic Stem Cell Differentiation and Early Embryonic Development

doi: 10.1371/journal.pone.0002814

Figure Lengend Snippet: (A) The Bcor Neo/Y and Bcor Gt/Y 129 strain derived ES cells can contribute to the B and T lineages. White blood cells from the spleens of Bcor Neo/Y and Bcor Gt/Y and appropriate controls were stained with antibodies to the 129 strain specific surface antigen Ly9.1 and the T cell markers CD4 and CD8 and B cell marker CD19. Cells were analyzed by flow cytometry and results are displayed as density plots with black boxes gating T or B cell populations of 129 origin. B6 and 129 strain wild type mice control for Ly9.1 specificity. CJ7 and E14 129 mouse strain derived ES cells were used to generate control chimera 1 (Con. Ch. 1) and control chimera 2 (Con. Ch. 2). Flow cytometry analyses were performed on chimeric control mice and on three mice for each mutant chimeric line, of which an example scatter plot is displayed for each. (B) Compared to chimeric controls, Bcor Neo/Y and Bcor Gt/Y 129 strain derived ES cell contribution to B and T cells is reduced. Graphs of scatter plot data show 129 strain contributions to each hematopoietic lineage. Black bars indicate the average contribution of control chimeric mice 1 and 2 (Con, black +) and the combined average of three Bcor Neo (Neo, red +) and three Bcor Gt (Gt, blue +) mice. Contribution to germinal center B cells is also reduced compared to controls as indicated by the final plot showing the PNA+ (peanut agglutinin; germinal center marker) percentage of CD19+ 129 derived B cells. P values were ≤0.05 for all hematopoietic cell populations tested (CD4; 0.004, CD8; 0.034, CD19; 0.023 and PNA; 0.001). (C) Bcor Neo/Y and Bcor Gt/Y ES cells can contribute to adult erythrocytes in chimeric mice but contribution is reduced in comparison to controls. Coomassie stained gel of hemolysates from erythrocytes of mice described above in panels A and B. Bcor Neo and Bcor Gt chimeric mice (Neo 1, 2 and 3 and Gt 1, 2 and 3) primarily express B6 derived ß -single globin where as chimeric control mice 1 and 2 primarily express 129 derived ß -major and ß -minor globin. The hemoglobin control lane (Hbb Con) was prepared from samples that contain ß -single, ß -major and ß -minor globin.

Article Snippet: Cells were stained with a 1∶200 dilution of appropriate primary antibody or lectin (Anti-Ly9.1-FITC (BD Pharmingen), Anti-CD19-PE (eBioscience), Anti-CD4-PerCP (BD Pharmingen), Anti-CD8-APC (eBioscience) and PNA-biotin (BD Pharmingen)) in FACs buffer for 20 minutes at room temperature and washed 1 time with 200 uL of FACs buffer.

Techniques: Derivative Assay, Staining, Marker, Flow Cytometry, Mutagenesis