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Blood, Vol. 109, Issue 10, 4280-4287, May 15, 2007
Defining the in vivo function of Siglec-F, a CD33-related Siglec expressed on mouse eosinophils
Blood Zhang et al.
109: 4280
Supplemental materials for: Zhang et al, Vol 109, Issue 10, 4280-4287
Files in this Data Supplement:
- Document 1. Supplemental methods (PDF, 102 KB)
- Table S1. Sequences of polymerase chain reaction (PCR) primers (PDF, 55.2 KB)
- Figure S1. Generation of Siglec-F−/− mice (JPG, 122 KB)
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(A) Strategy for siglecf gene targeting. Neo indicates neomycin-resistance gene; tk, thymidine kinase gene; closed box, siglecf exon; and arrowhead, loxP site. Positions of exons 5 and beyond are not shown. The XhoI site at the end of long arm was destroyed during targeting vector preparation and is indicated in parentheses. A BsmI site between exons 3 and 4 was used in the vector preparation (not shown). Schematic representation of WT, siglecfflox + neo-tk, siglecfflox - neo-tk, and null alleles is indicated below. BamHI, HindIII, XhoI, and XbaI sites in the genomic DNA regions are indicated with B, H, Xh, and Xb, respectively. XhoI, BamHI, and XbaI sites introduced during targeting vector preparation were indicated with italics. Positions and names of primers used in PCR screening are indicated. Position of the DNA probe (5′ XbH) used in Southern blot analyses is also shown. (B) Southern blot analysis of embryonic stem (ES) cell DNA. Genomic DNA prepared from ES cells was digested, blotted, and probed as described in Supplemental Methods. (i) G418-resistant ES cells were screened for homologous recombinants by PCR (PCR-1), and DNA samples of candidate clones were analyzed by Southern blotting. (Left) Nonhomologous recombinant with both wild-type alleles intact; (right) homologous recombinant ES clone with one wild-type and one siglecfflox + neo-tk allele. (ii) Gancyclovir-resistant ES cells were screened for neo-tk cassette removed yet floxed siglef allele-intact (siglecfflox - neo-tk/+) clones by PCR (PCR-3), and candidate ES DNA was analyzed by Southern blotting. (Left) Parental ES cell clone with one wild-type and one siglecfflox + neo-tk allele. (Right) ES cell clone with one wild-type and one siglecfflox - neo-tk allele. (C) PCR was performed on toe DNA isolated from offspring of siglecf+/− intercrosses. This PCR reaction gives a 1266-bp fragment from WT allele and a 364-bp fragment for the mutant allele. (D) Peripheral blood leukocytes were stained with anti–Siglec-F and anti-CCR3, and analyzed by flow cytometry. Siglec-F–null mice have peripheral eosinophil numbers in the normal range, with normal expression of eosinophil marker CCR3, but no Siglec-F expression.
- Figure S2. Antibody-mediated cross-linking of Siglec-F induces eosinophil apoptosis (JPG, 27 KB)
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Peripheral blood eosinophils from IL-5 transgenic mice were incubated in vitro and the level of apoptosis was determined as described in “Materials and methods.” While anti–Siglec-F antibody alone ( Siglec-F; gray bar) did not induce apoptosis above background (No Ab; white bar), the addition of a secondary cross-linking antibody (Siglec-F + 2′Ab; black bar) significantly enhanced apoptosis. ***P < .001. Note that the background level of apoptosis is high because of the withdrawal from IL-5.
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