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A genomic DNA fragment of 11 kb encompassing exons 1 through 8 of the mouse PI3Kγ was used to integrate the IRES-LacZ cassette followed by the PGK-neomycin resistance gene from the pWH9 plasmid (provided by R. Fässler, Lund University, Sweden) in exon 2, 105 base pairs downstream of the first coding ATG. Five independently targeted R1 ES cell clones were identified by Southern blot hybridization. No evidence for random integration was detected (17).
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Animal experiments were carried out in accordance with institutional guidelines
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All ES cells clones were injected into C57BL/6 blastocysts. For genotyping of mice, DNA derived from tail biopsies was amplified by polymerase chain reaction with two primers sets (1: 5′-GGAGAACTAT-GAACAACCGG-3′, 5′-CAACTTCCAGTAATGCAGGC-3′; 2: 5′-CTGCTCTTTACTGAAGGCTC-3′, 5′-CAACT-TCCAGTAATGCAGGC-3′) that detect the WT and targeted allele, respectively. Phenotypic analysis was performed on two lines derived from independent clones, and results were confirmed in a 129sv-C57BL/6 mixed and 129sv inbred genetic background. The IRES-LacZ reporter gene under the control of the PI3Kγ promoter was expressed in peripheral blood leukocytes and in spleen macrophages [(17); for expression studies see H. G. Bernstein, G. Keilhoff, M. Reiser, S. Freese, R. Wetzker, Cell Mol. Biol. 6, 973 (1998)]. Animal experiments were carried out in accordance with institutional guidelines.
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0343813229
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note
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2 in Iscove's modified Dulbecco's medium (IMDM medium, Life Technologies). Macrophages were obtained by washing the peritoneal cavity of mice 5 days after intraperitoneal injection of 1 ml of 3% thioglycollate (Difco). Antibodies to PI3K were from A. Klippel (Berlin, Germany; PI3Kα), R. Wetzker (Jena, Germany; PI3Kγ), B. Vanhaesebroeck (London, UK; PI3Kδ), and St. Cruz Biotechnology (PI3Kβ).
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IL-8 (50 nM) stimulated a significant [P < 0.05, analysis of variance (ANOVA)] increase in adhesion from 27.1% (SE = 4.8%, n = 10; percentage of total administered cells) to 43.5% (SE = 6.0%, n = 14) in WT, but not in PI3Kγ-null neutrophils [unstimulated, 27.8 ± 3.3% (n = 10) versus 27.0 ± 2.9% (n = 14) in the presence of IL-8]. C5a- and fMLP-induced adhesion were not significantly reduced by the loss of PI3Kγ. Teflon-coated 12-well glass slides (Marienfeld) were coated with fibronectin (20 μg/ml; Sigma) solution. Calcein-AM (Molecular Probes)-loaded PMNs (20 μl) were applied to the glass slides. After stimulation, nonadherent cells were removed by washing. Fluorescence of attached cells was measured in a Bio-Tek FL600 fluorescence plate reader (excitation, 485 nm, 20-nm slit; emission, 530 nm, 25-nm slit).
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We thank L. Barberis, M. F. Brizzi, G. Bulgarelli-Leva, G. Frascaroli, W. Luini, G. Montrucchio, M. Laffargue, and R. Calvez for their support and assistance. We also thank G. Tarone, P. Defilippi, L. Fumagalli, and G. Topley for critically reading the manuscript; B. Vanhaesebroeck, R. Wetzker, A. Klippel, and B. Hemmings for antibodies; C. Dahinden for C5a; and B. Moser for chemokines. Supported by the Progetto Finalizzato Biotecnologie, CNR (project biotechnology), Associazione Italiana per la Ricerca sul Cancro (AIRC), San Salvatore Foundation, Telethon grant E 635, and Swiss National Science Foundation grant 31-50506.97.
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