Supporting information for Humbles et al. (2002) Proc. Natl. Acad. Sci. USA 99 (3), 1479–1484. (10.1073/pnas.261462598)
Supporting Methods
Targeted Disruption of the CCR3 Gene.
Mouse 129/sv genomic DNA library was screened with mouse CCR3 cDNA. A 10-kb genomic fragment including the CCR3 open reading frame was used to construct the targeting vector. A 3-kb HinDIII fragment containing the start codon and part of the N-terminal coding sequence for the receptor (39 base pairs) and a 5' untranslated region was deleted and replaced by a neomycin-resistance cassette under the control of the phosphoglucokinase promoter (PGK-neo). This mutated fragment was subcloned into the pPNT vector for double selection with geneticin and gancyclovir. The targeted vector was linearized and electroporated into J1 embryonic stem cells. Doubly resistant clones were screened by Southern analysis, and two correctly targeted clones were selected for blastocyst injection into BALB/c mice. Chimeric males were bred to yield germline transmission of the targeted allele. Heterozygote (+/-) animals were then bred with wild-type BALB/c mice to yield second- and third-generation BALB/c backcrossed mice. Heterozygote animals from these matings were then bred against each other to yield homozygote CCR3-/- and wild-type littermates, which were used for all studies described. All experiments were subsequently repeated and reproduced in seventh-generation BALB/c backcrossed mice.RT-PCR Analysis.
Total RNA was prepared from mouse bone marrow by using Ambion (Austin, TX) RNAqueous reagents. RT-PCR was performed by using 200 ng of DNAse I-treated RNA following Titan one tube RT-PCR system protocol (Boehringer Mannheim). The primer sequences were as follows: CCR3 forward, 5'-ATGGCATTCAACACAGAT-3', reverse, 5'-ATCTGTCAATTGTCAGCA-3' (approximately 400-bp product); CXCR3 forward, 5'-ATGTACCTTGAGGTTAGT-3', reverse, 5'-AGTTGTACTGGCAATGGG-3' (approximately 600-bp product).Calcium Mobilization
. CCR3-deficient and wild-type littermates were backcrossed into IL-5 transgenic mice (1). Presence of the IL-5 transgene in offspring was determined by Southern blot analysis (not shown). White blood cells from IL-5/CCR3 wild-type and IL-5/CCR3-/- mice were obtained as previously described (2). Cells were washed twice and loaded with Fura-2 AM by incubating at 37°C for 45 min in 120 mM NaCl containing 25 mM Hepes, pH 7.5, 1 mM CaCl2, 5 mM MgCl2, and 2 mg/ml Fura-2 AM (Molecular Probes). Cells were washed twice in the same buffer and resuspended at 5 ´ 106/ml; approximately 30% of the cells were eosinophils, as determined by cytospin analysis. Intracellular calcium concentration was determined after addition of ligands monitoring the relative fluorescence with excitation at 340 and 380 nm and emission at 510 nm.Ribonuclease Protection Assay.
Lung tissue was stored overnight at 4°C in RNAlater, and the RNA was prepared by using the RNAqueous kit (Ambion, Austin, TX). Ribonuclease protection assays were performed by using the RiboQuant kit purchased from PharMingen. All procedures were performed according to the instructions provided by the manufacturers.References: