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We generated a null VEGFR-3 allele by introducing a LacZ cassette into the VEGFR-3 locus (11). The 3-kb Kpn I-Not I and Bam HI-Kpn I fragments from 129SV mouse genomic DNA library (λFIX II; Stratagene) were used as the arms of the targeting vector (Fig. 1). The gene-targeting event was confirmed by Southern (DNA) blot analysis (Fig. 1) (D. J. Dumont et al., data not shown), and two independent embryonic stem cell lines were used to make heterozygous mice through blastocyst injection. Wild-type, heterozygous, and homozygous embryos and mice were identified by Southern blot analysis of yolk sac and tail DNA, respectively.
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Embryos were stained for β-Gal (12), photographed, and subsequently dehydrated and embedded in paraffin. Seven-micrometer sections were stained immunohistochemically for PECAM-1 (Pharmingen, San Diego, CA) with TSA Kit (NEN, Boston, MA). Mouse VEGFR-3 [base pairs (bp) 22 to 214; GenBank L07296] and VEGF-C (bp 499 to 656; GenBank U73620) cDNA probes were used for in situ hybridization (9).
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We thank A. Kaipainen for helpful discussions, E. Koivunen for expert help with the mice, and M. Helanterä, P. Ylikantola, and T. Tainola for technical assistance. Supported by research grants from the Medical Research Council (Canada) and AMGEN (to D.J.D.) and by the Finnish Cancer Organizations, the Finnish Academy, the Sigrid Juselius Foundation, the Finnish State Technology Development Centre, and the European Union Biomed Program.
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