Establishment of a transgenic cell line stably expressing human cytochrome P450 2C18 and identification of a CYP2C18 clone with exon 5 missing

Figure 1 Scheme of recombinants of pGEM-CYP2C18 and pREP9-CYP2C18

Figure 2 Electrophoresis identification of recombinants of pGEM-CYP2C18 and pREP9-CYP2C18. Lane 1: Marker (λ/EcoR I and Hind III); 2: pGEM-T vector; 3: Recombinant of pGEM-CYP2C18 digested by Nhe I and Xho I (incompleted digestion); 4: PCR products of CYP2C18 (1.67 kb); 5: Recombinant of pREP9-CYP2C18 digested by Nhe I and Xho I; 6: pREP9 vector.

Figure 3 Electrophoresis and sequencing identification of a CYP2C18 clone with exon 5 missing. A: Electrophoresis identification of a CYP2C18 clone with an exon 5 being skipped; Lane 1: Marker (λ/EcoR I and Hind III); 2: Recombinant of pGEM-CYP2C18 digested by Nhe I and Xho I (incompleted digestion); B: Partial sequencing of an CYP2C18 cDNA clone with an exon 5 being skipped. The upper sequence represent the sense strand and the underside sequence represent the anti-sense strand being sequenced.

Figure 4 Representative chromatogram of extracts. A Shim-pack CLC-ODS column (15 × 0.6 cm i.d.) was used. The mobile phase was constituted with 0.05% phosphoric acid (pH2.6), acetonitrile (6:4/V:V) with the flow rate of 1 mL. min^-1. Hydroxytolbutamide was monitored at 230 nm. A: hydroxytolbutamide; B: tolbutamide