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AIM: To study the enzyme activity of CYP2C18 variant with exon 5 skipped. METHODS: A full length CYP2C18 cDNA X1 and an exon 5 skipped variant CYP2C18 X2 were separately subcloned into mammalian expression vector pREP9 to transfect HepG2 cells. The expression of CYP2C18 mRNA in transgenic cells and human liver tissues were determined by RT-PCR. The enzyme activity of CYP2C18 to oxidate tolbutamide in postmitochondrial supernate (S9) fraction was determined by HPLC. The cytotoxicity of ifosfamide to transgenic cells was evaluated by MTT test. RESULTS: HepG2-CYP2C18 X1 cells showed strong expression of the full length CYP2C18 mRNA. On the other hand, HepG2-CYP2C18 X2 cells had only infinitesimal expression of the exon-skipped CYP2C18 as well as the full length CYP2C18, while non-transfected HepG2 cell only demonstrated an infinitesimal expression of the full length CYP2C18. The expression of CYP2C18 exons 2 to 7 was also analyzed by RT-PCR in 7 extratumoral liver tissues. Among them, 3 samples expressed on
AIM: To study the enzyme activity of CYP2C18 variant with exon 5 skipped. METHODS: A full length CYP2C18 cDNA X1 and an exon 5 skipped variant CYP2C18 X2 were separately subcloned into mammalian expression vector pREP9 to transfect HepG2 cells. The expression of CYP2C18 mRNA in The cytotoxicity of ifosfamide to transgenic cells was evaluated by MTT test. RESULTS: The enzyme activity of CYP2C18 to oxidate tolbutamide in postmitochondrial supernate (S9) fraction was determined by HPLC. The cytotoxicity of ifosfamide to transgenic cells was evaluated by RT- CYP2C18 X1 cells showed strong expression of the full length CYP2C18 mRNA. On the other hand, HepG2-CYP2C18 X2 cells had only infinitesimal expression of the exon-skipped CYP2C18 as well as the full length CYP2C18, while non-transfected HepG2 cell only demonstrated The expression of CYP2C18 exons 2 to 7 was also analyzed by RT-PCR in 7 extratumoral liver tissues. Among them, 3 s amples expressed on