Figure 9.

Expression of normal and mutant C₂₇ 3β-HSD genes in cultured HEK 293 cells. The C₂₇ 3β-HSD gene was amplified using PCR from the genomic DNA of either a normal subject (Normal) or an individual with intrahepatic cholestasis (MU2) and inserted into the plasmid pCMV6 as described in Methods. The resulting expression vectors were introduced together with a plasmid expressing the mouse CYP7B1 oxysterol 7β-hydroxylase cDNA (pCYP7B1) into HEK 293 cells by cotransfection. After 24 hours, the ability of the transfected cells to convert [³H]cholest-5-ene-3β,24-diol (24-hydroxycholesterol, lanes 1–4), [³H]cholest-5-ene-3β,25-diol (25-hydroxycholesterol, lanes 5–8), and [³H]cholest-5-ene-3β,27-diol (27-hydroxycholesterol, lanes 9–12) was determined by thin-layer chromatography. The chromatogram was exposed to x-ray film for 6 days.