Engineering of the loxP-<i>Itga2</i> transgene.

<p><b>A</b>) The targeting vector construction is depicted schematically to show positions of the key restriction sites used to clone exon1 flanked by loxP sites (middle), long arm (left) and short arm (right) DNA segments into the vector pBS-FRT-neo-FRT, which already contained the Neomycin-Kanamycin resistance marker flanked by FRT sequences, removable by Flp-recombinase. The positions of loxP and FRT sites are shown relative to exon 1 (E1). A unique Hind 3 restriction site was introduced next to the loxP sequence to facilitate the verification of a correct homologous recombination event in later stages. <b>B</b>) The screening strategy to identify ES cells with a correct recombination event. Two PCR reactions were employed, each utilizing one primer located within the natural α2 gene sequence beyond the end of the short (P8) or long (P9) arms, thus excluding amplification of random insertions. <b>C</b>) Genotyping strategy employed to detect removal of the Neomycin cassette by Flp-recombinase using primer pairs P12/P13 and P14/P15. Once homozygous mice were obtained, the correct location of the trans-gene was verified by PCR amplification of the entire region using primer pair P9/P11, each located in the natural <i>Itga2</i> gene sequence beyond the long and short arms, followed by <i>Hind3</i> restriction fragment length polymorphism analysis. Cre-recombinase mediated removal of the floxed exon 1 was confirmed by PCR using primer pair P12/P15.