Genomic DNA was purified from sorted B220+ CD43? bone marrow cells of 3-wk-old nontransgenic or Blk(Y495F) transgenic mice. but present in all transgenic arrays or (b) present in all control arrays but absent from all transgenic arrays. A gene was included in the third arranged if its normalized manifestation differed by more than fivefold between transgenic and nontransgenic samples. Hierarchal clustering was performed using the standard correlation coefficient like a range metric. Online Supplemental Material. Fig. S1 health supplements Fig. 2 and shows the effect of Blk (Y495F) on manifestation of the B cell developmental markers CD24, CD25, CD2, and c-kit in RAG-deficient or Mt/MT mice, as assessed Saterinone hydrochloride by fluorescence cytometry. Table S1 shows the distribution of B220 CD43+ and B220 CD43? cells in the bone marrow of RAG-deficient or MT/MT mice expressing the Blk (Y495F) transgene. Table S2 shows the distribution of B220 IgM+ and B20 IgM? B cells in the spleens of Blk (Y495F) transgenic mice. Table S3 health supplements Fig. 6 C and assigns the in a different way indicated genes of known function to practical groups. Table S4 depicts the primer pairs utilized for RT-PCR, whose results are demonstrated in Fig. 6 D. Fig. S1 and Furniture S1CS4 are available at http://www.jem.org/cgi/content/full/jem.20030729/DC1. Open in a separate window Number 2. Manifestation of Blk(Y495F) circumvents developmental blocks in RAG-deficient or MT/MT mice. (A) Bone marrow cell suspensions were prepared from 3C5-wk-old RAG-2?/? mice bearing the Blk(Y495F) transgene (right), or from age-matched, nontransgenic RAG-2?/? littermates (left). Cells were stained with an anti-B220 antibody and Saterinone hydrochloride counterstained with antibodies for more surface markers as indicated. Figures show percentages of cells in the related quadrants. (B) Analysis, as with A, of bone marrow cells from 3C5-wk-old MT/MT mice bearing Blk(Y495F) transgene (ideal), or from age-matched, nontransgenic MT/MT littermates (left). Open in a separate window Number 6. Focuses on of proximal and distal signaling in B cell progenitors from Blk(Y495F) transgenic mice. (A) Constitutive tyrosine phosphorylation of Ig in transgenic B cell progenitors. Lysates were prepared from proCB cells of nontransgenic (lanes 1, 4, and 7) or transgenic (lanes 2, 5, and 8) mice, and Ig was immunoprecipitated (lanes 4C8). Control immunoprecipitations were performed from a thymocyte lysate (lanes 3 and 6) or with nonimmune IgG (lanes 1C3). Undiluted (lanes 1C6) and twofold diluted (lanes 7 and 8) immunoprecipitates were fractionated by electrophoresis alongside whole cell lysates (lanes 9C11). Phosphotyrosine (top) and Ig- (bottom) were recognized by sequential immunoblotting. Arrows mark the position of Ig. (B) Constitutive tyrosine phosphorylation of Syk in transgenic B cell progenitors. Lysates were prepared from nontransgenic (lanes 1 and 2) or transgenic (lanes 3 and 4) proCB cells as with A. Syk was immunoprecipitated (lanes 2 and 4) and control immunoprecipitations were performed with nonimmune IgG (lanes 1 and 3). Phosphotyrosine (top) and Syk (bottom) were recognized by sequential immunoblotting. (C) Differential gene manifestation in proCB cells from transgenic and nontransgenic mice. 35 genes of known function that were differentially indicated in nontransgenic (remaining) and transgenic (right) proCB cells Saterinone hydrochloride are demonstrated. Each column corresponds to one microarray. Red represents manifestation above and green represents manifestation below the median value. Black represents manifestation in the median and gray represents no detectable manifestation. (D) Confirmation of differential manifestation. Total RNA from nontransgenic (lanes 1C4) or transgenic Saterinone hydrochloride (lanes 5C8) proCB cells was reverse transcribed, diluted serially fourfold, and used like a template for amplification of the transcripts indicated at right. Products were fractionated by gel electrophoresis and recognized with ethidium bromide. Results Development of B Cell Progenitors in Bone Marrow of Blk(Y495F) Transgenic Mice. We 1st considered whether active Blk might deliver proliferative signals self-employed of pre-BCR manifestation using a line Rabbit polyclonal to TUBB3 of transgenic mice, Blk(Y495F)-15, in which a constitutively active Blk mutant is definitely indicated specifically in the B lymphoid lineage (26)..