Molecular Dissection of B-lymphocyte Signalling Using Expression Profiling

Purpose: Bruton’s tyrosine kinase is crucial for B-lymphocyte development. By the use of gene expression proWling, we have iden-tiWed four expressed sequence tags among 38 potential Btk target genes, which have now been characterised.Methods: Bioinformatics tools including data mining of additional unpublished gene expression proWles, sequence veriWcation ofPCR products and qualitative RT-PCR were used. Stimulations targeting the B-cell receptor and the protein kinase C were used toactivate whole B-cell splenocytes.Results: Target genes were characterised as Lim domain only 7 (Lmo7); Myosin1e (Myo1e); SAM and SH3 domain containing 1(Sash1); and Mucolipin2 (Mcoln2). Expression was found in cell lines of diVerent origin and developmental stages as well as in wholeB-cell splenocytes and Transitional type 1 (T1) splenic B-cells from wild type and Btk-defective mice, respectively. By the use of semi-quantitative RT-PCR we found Sash1 not to be expressed in the investigated haematopoietic cell lines, while transcripts were foundin whole splenic B-cells from both wild type and Btk-defective mice, whereas Lmo7, Myo1e, and Mcoln2 were expressed in both B-cell lines and primary B-lymphocytes. Except for Lmo7, the transcript level was similarly aVected by stimulation in control and Btk-defective cells. 2005 Elsevier Inc. All rights reserved.