To examine further whether the ESE in exon 2 of CD200 determined the fate of the exon (inclusion or exclusion), site-directed mutagenesis was performed to mutate the ESE element in the alternative splicing construct, replacing the ESE (TCCTGA) with a restriction enzyme BsiWI site (CGTACG) ( Figure 3C ) or to delete the ESE. After characterizing the mutation or deletion construct by sequencing, the splicing construct was transfected to Daudi and SK-N cells. Total RNA was extracted from cells 48 h after transfection and real-time RT-PCR was carried out. As shown in Figures 3C and D, and 4A and B, expression of the full-length transcript (exon 2 inclusion) was reduced in both Daudi and SK-N cells after mutation or deletion of the ESE in exon 2. These data suggest that the ESE in exon 2 of CD200 promotes exon 2 inclusion.