An ESE site was identified in exon 2 of the human CD200 using computational methods RESCUE-ESE (34) and ESEfinder (35) . To mutate the ESE site, site-directed mutagenesis was employed using QuickChange II XL site-directed mutagenesis kit from Stratagene. Two mutagenic primers were synthesized, in which the ESE site was replaced by a BsiWI site or deleted, and purified by polyacrylamide gel electrophoresis (PAGE). The sequences of the primers used are shown in Table 1 (the mutated region was underlined). The mutagenesis reaction was carried out in 50 ml total volume with 40 ng of template DNA, 125 ng of each primer and 2.5 U PfuUltra high-fidelity (HF) DNA polymerase and 3 ml of QuickSolution reagent provided by Stratagene. The cycling conditions included a 1-min initial denaturation at 95 C, 18 cycles with 50 s denaturation at 95 C, 50 s annealing at 58 C and 40 min extension at 68 C, and a final extension of 7 min at 68 C. The product was then subjected to digestion with 10 U of DpnI for 2 h at 37 C, selectively removing the parental, methylated, and nonmutated strands. Four microliters of DpnI-treated DNA was then transformed into XL10-Gold Ultracompetent cells. Cells were plated and incubated for selection of ampicillin-resistant clones. Ten isolated ampicillin-resistant clones were picked at random and their mutated or deleted regions were characterized by DNA sequencing.