To gain insight into the role of the ESE in exon 2 of CD200, we generated an alternative splicing minigene construct containing the genomic region from exon 1 to exon 3 of the human CD200 ( Figure 2B) . A 12-kb fragment bearing this genomic region was characterized by sequencing and restriction enzyme digestion, and ligated to a pcDNA 3.0 expression vector. The construct was transfected independently to human B cell line Daudi and neuronal cell line SK-N. After 48 h, RNA was extracted from each cell population for detection of the exogenous expression of splicing pattern of CD200. RNA was also isolated from nontransfected Daudi and SK-N cells for detection of the endogenously expressed splicing pattern. To measure quantitatively the expression levels of the two splice variants, real-time RT-PCR was performed using the primer pairs located in different regions ( Figure 3A ). The specificity of the primers for amplification of full-length and truncated CD200 was examined. As shown in Figure 3B , the primer pair used for full-length CD200 did not amplify the template from CD200 RNA lacking exon 2 (truncated form), whereas the primer pairs for truncated CD200 were not able to amplify the template from CD200 RNA containing exon 2 (full-length form). Each primer pair generated only a single product (Supplementary Figure 1A) and the standard curves generated from each primer pair are parallel with slopes between À3.1 and À3.6 (Supplementary Figure 1B) . The exogenous expression of CD200/ CD200 tr had a similar pattern to the corresponding endogenous one in Daudi cells or SK-N cells ( Figure 3D and E).