However, all of these studies assessed only agonists’-inducible platelet reactivity

xpected frequencies of transmission of the floxed allele, and homozygous mice appeared healthy. Expression of PF-562271 calponin-3-GFP from the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19712481 endogenous Cnn3 locus To test whether the abovementioned targeting strategy indeed promotes the expression of calponin-3-GFP from the endogenous promoter, bone marrow-derived pre-B cells from homozygous knock-in mice were analyzed by RT-PCR. Using non-targeted pre-B cells as a control, we were able to confirm that exon 1 is indeed correctly spliced to the mini gene, giving rise to a transcript encoding full-length calponin-3-GFP. In correspondence with this, western blot analysis of the same cells showed a band at the same height as pre-B cells stably expressing a full-length calponin-3-GFP construct. Moreover, GFP fluorescence from the fusion construct was readily detected by flow cytometry in B cells isolated from the bone marrow of Cnn3 ki f/f mice, supporting the initial protein expression data. Beyond B cells, calponin-3-GFP expression was also clearly visible in Cnn3 ki f/f embryos using fluorescence imaging, showing a broad expression pattern throughout early development Fig 3. Calponin-3-GFP is expressed from the endogenous Cnn3 locus. A. RT-PCR analysis of the Cnn3 mRNA splicing in targeted cells. Total RNA from the pre-B cell line Oct as well as from pre-B cells derived from a Cnn3 ki f/f mouse was transcribed into cDNA and analyzed by PCR. Primer pairs for PCRs 1 and 2 are depicted in the schematic illustration of the targeted allele. A sample lacking cDNA served as a control. B. Western blot analysis for expression of the calponin-3-GFP fusion protein from the targeted Cnn3 locus. Pre-B cells derived from a Cnn3 ki f/f mouse or a non-targeted littermate were lysed and subjected to SDS-PAGE and blotting. Oct pre-B cells expressing GFP and calponin-3-GFP were used as a control. Equal loading was demonstrated by antieIF4. C. Flow cytometric analysis of cells from a Cnn3 ki f/f mouse or a non-targeted +/+ littermate. Cells were isolated from the bone marrow of the respective mice, stained with an anti-IgM antibody and analyzed for expression of IgM and GFP by flow cytometry. Numbers indicate the percentage of cells in the respective gate. doi:10.1371/journal.pone.0128385.g003 8 / 16 Calponin-3 in B Lymphocyte Development . Together, these data suggest that the knock-in mouse model is suitable for monitoring calponin-3 expression in different tissues and in response to different stimuli. Calponin-3 is expressed throughout B cell development, but is restricted to a subset of thymic T cells In order to determine the level of calponin-3 protein expression in the different developmental subpopulations of B cells, we analyzed cells isolated from the bone marrow, the spleen, lymph nodes and the peritoneal cavity of Cnn3 ki f/f and control mice, respectively, by flow cytometry. Using the change in GFP mean fluorescence intensity as a read out, our reporter mouse revealed a clear expression of calponin-3-GFP already in pro-/pre-B cells in the bone marrow, with even higher levels in immature and mature B cells. In contrast, non-B cells showed no calponin-3-GFP fluorescence, recapitulating the western blot analysis. In the spleen, calponin-3 expression was equally high in splenic transitional 1 and follicular B cells, with only the pool of marginal zone PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19710274 and transitional 2 B cells appearing slightly lower in GFP fluorescence. In the periphery, B cells isolated from lymph nodes retained high calponin-3 levels,