Fig. 1.
Fig. 1. Targeted disruption of the mouse DARC gene. / (A) mDARC targeting strategy. Top line: Endogenous DARC gene locus showing coding exon I and exon II in a 9-kb EcoRI fragment. A 3′ 1-kb SpeI-EcoR1 located outside the targeting construct was used as a probe for Southern blot analysis. Primers D1 and D2 were used for PCR identification of the wild-type DARC gene, and primers D2 and D3 were used to detect DARC cDNA by RT-PCR analysis. Middle line: The targeting plasmid, a 1.1-kbneo gene, was inserted to replace theBglII-XhoI fragment. This eliminates a part of the intron and 90 bp of exon II. A 5′ 4.3-kbEcoRI-BglII fragment was used as the 5′ arm of homology and a 3′ 1.2-kb XhoI-PstI fragment containing most of the coding exon was used for the 3′ homology. A TK gene was inserted for negative selection of ES cells. The OSDUPDEL vector was used to create the targeting construct. Bottom line: The correctly targeted allele. There is a unique EcoRI site introduced with the neo gene that results in a reduction of the germline 9-kb EcoRI fragment to 4.5 kb when the DARC probe is used for Southern blot analysis. Alternatively, the correctly targeted allele can be detected by PCR analysis by using primers D2 and Dneo−. (B) Southern blot showing correct targeting at the DARC locus. Genomic DNA prepared from the tails of F2+/+, F2+/−, and F2−/− mice were digested with EcoRI and probed with the DARC probe, detecting bands of 9.0 kb (▸, wild-type) and 4.5 kb (▹, targeted). (C) PCR gel showing correct targeting at the DARC locus. Genomic tail DNA from F2+/+, F2+/−, and F2 −/− mice was analyzed by PCR analysis with the use of primers D1, D2, and Dneo, resulting in a 400-bp wild-type product (▸) and a 300-bp targeted product (▹). (D) RT-PCR analysis of DARC-deficient mice. Total RNA was isolated and cDNA was obtained from the spleens of F2 DARC+/+, F2 DARC +/−, and F2 DARC −/− mice. There was no observable difference in the expression of DARC mRNA (▸) between +/+ and+/− mice; however, we did observe an alternate splicing product that is produced in the spleen at 800 bp. GAPDH mRNA (▹) served as a control. There is a faint 1.4-kb band that results from a nonfunctional message that is produced across the neoinsert. This RT-PCR analysis indicates that DARC−/− mice have no normal DARC mRNA in their spleens. (E) Absence of IL-8 binding to DARC-deficient RBCs. For a competitive binding assay, RBCs were isolated from F2 DARC+/+, +/−, and−/− mice and incubated with 0.5 nmol/L of125I-labeled IL-8 and varying amounts of unlabeled IL-8 (0, 100, or 500 nmol/L). Each data point represents the average of duplicate samples. Similar results were obtained in 3 independent experiments. (F) Absence of MCP-1 binding to DARC-deficient RBCs. Competitive binding assays with 125I-MCP-1 were performed as described above. Similar results were obtained in 2 independent experiments.

Targeted disruption of the mouse DARC gene.

(A) mDARC targeting strategy. Top line: Endogenous DARC gene locus showing coding exon I and exon II in a 9-kb EcoRI fragment. A 3′ 1-kb SpeI-EcoR1 located outside the targeting construct was used as a probe for Southern blot analysis. Primers D1 and D2 were used for PCR identification of the wild-type DARC gene, and primers D2 and D3 were used to detect DARC cDNA by RT-PCR analysis. Middle line: The targeting plasmid, a 1.1-kbneo gene, was inserted to replace theBglII-XhoI fragment. This eliminates a part of the intron and 90 bp of exon II. A 5′ 4.3-kbEcoRI-BglII fragment was used as the 5′ arm of homology and a 3′ 1.2-kb XhoI-PstI fragment containing most of the coding exon was used for the 3′ homology. A TK gene was inserted for negative selection of ES cells. The OSDUPDEL vector was used to create the targeting construct. Bottom line: The correctly targeted allele. There is a unique EcoRI site introduced with the neo gene that results in a reduction of the germline 9-kb EcoRI fragment to 4.5 kb when the DARC probe is used for Southern blot analysis. Alternatively, the correctly targeted allele can be detected by PCR analysis by using primers D2 and Dneo−. (B) Southern blot showing correct targeting at the DARC locus. Genomic DNA prepared from the tails of F2+/+, F2+/−, and F2−/− mice were digested with EcoRI and probed with the DARC probe, detecting bands of 9.0 kb (▸, wild-type) and 4.5 kb (▹, targeted). (C) PCR gel showing correct targeting at the DARC locus. Genomic tail DNA from F2+/+, F2+/−, and F2 −/− mice was analyzed by PCR analysis with the use of primers D1, D2, and Dneo, resulting in a 400-bp wild-type product (▸) and a 300-bp targeted product (▹). (D) RT-PCR analysis of DARC-deficient mice. Total RNA was isolated and cDNA was obtained from the spleens of F2 DARC+/+, F2 DARC +/−, and F2 DARC −/− mice. There was no observable difference in the expression of DARC mRNA (▸) between +/+ and+/− mice; however, we did observe an alternate splicing product that is produced in the spleen at 800 bp. GAPDH mRNA (▹) served as a control. There is a faint 1.4-kb band that results from a nonfunctional message that is produced across the neoinsert. This RT-PCR analysis indicates that DARC−/− mice have no normal DARC mRNA in their spleens. (E) Absence of IL-8 binding to DARC-deficient RBCs. For a competitive binding assay, RBCs were isolated from F2 DARC+/+, +/−, and−/− mice and incubated with 0.5 nmol/L of125I-labeled IL-8 and varying amounts of unlabeled IL-8 (0, 100, or 500 nmol/L). Each data point represents the average of duplicate samples. Similar results were obtained in 3 independent experiments. (F) Absence of MCP-1 binding to DARC-deficient RBCs. Competitive binding assays with 125I-MCP-1 were performed as described above. Similar results were obtained in 2 independent experiments.

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