E. to develop a and double-knockout mouse (genes and the use of these mice in complement-sufficient (and double knockout mouse, we undertook a targeted deletion strategy (Fig. 1a), because the very short genetic (~0.005 cM) and physical (~10 kb) range between the genes practically precluded a recombination-dependent (and genes indicated that this region, which would be also deleted by our construct, did not contain either known genes or expressed sequence tags (EST). Southern blotting confirmed that homologous recombination for both arms of the focusing on cassette occurred in two self-employed 129 Sera cell lines that were Edonerpic maleate selected for microinjection into B6 blastocytes (Fig. 1b). Producing chimeras crossed with B6 mice yielded and genes was confirmed by (1) Northern analysis with and mRNA transcripts in multiple cells, and (2) FACS analysis with specific anti-mCd59a and mCd59b monoclonal antibodies showing the physical absence of both mCd59 proteins in erythrocytes (data not demonstrated) as reported in Ref. 17 and in platelets of several and exon 1 and exon 2 of from the gene. Double-headed arrows show the size of the fragments seen in Southern blot analysis of wildtype (top) and targeted (bottom) alleles after the external probe (blue package) hybridized to Hind III digestion of genomic DNA. Black rectangles: exons; reddish rectangles: exons. (b) Southern analysis with external probe (blue square demonstrated in Fig. 1a) to determine the homologous recombination of remaining arm. The 20, 17, and 13 kb Hind III bands represent expected wild-type, targeted fragment, and nonspecific binding fragment, respectively. (c) (d) or (e) mRNA manifestation in multiple cells Edonerpic maleate of (d) or (e) cDNA probe, and bottom panel is loading control. F, excess fat cells; T, testes; K, kidney; L, liver; H, heart; B, brain. Open in a separate window Number 2 The deficiency of mCd59a and mCd59b protein in the platelets of mCd59ab?/? mice. (a) The predominance of platelets in the cell populace analyzed by FACS was confirmed with an antibody specific for the platelet marker Edonerpic maleate CD41. (b) FACS analysis of the mCd59a or mCd59b proteins in platelets from = 4)506.7 44.210.4 1.3*1.6 0.2*= 4)589.7 99.50.3 0.50.18 0.3 Open in a separate window Actual fluorences density unit (FDU) equals FDU acquired by main antibody and FITC-conjugated secondary antibody minus FDU acquired by FITC conjugated secondary antibody. Data are displayed by Mean SEM. * 0.05 vs. genes (Fig. 3e). This improved level of sensitivity of 0.05 vs. (Table II, and Fig 4aCe). In the real B6 genetic background of the mice used for this study, 35% of the 0.01, = 12. (d) Percentage of anemic mice (Hb 13g/dl at 2C4 month of age): Adamts5 = 30), = 34) and = 14). We compared the proportions in the extremes of the distributions using Fishers precise test as explained in21. * 0.01 vs. 0.001, vs. = 34)14.7 0.14= 28)14.7 0.16a= 30)13.6 1.0b= 14)14.2 0.22c Open in a separate window *Mice are 2C4 months aged. aComparison between 0.1. bComparison between 0.01. Edonerpic maleate cComparison between 0.05. Progressive loss of male fertility in 0.0001 vs. and double- knockout mice and (2) the phenotypic characterization of or double-knockout mice Both mouse genes have four exons; together they span 45.6 kb in the mouse genome: 19 kb was amplified using a bacterial artificial chromosome (BAC) that covers.