DHCR24^−/− cells adapted to serum-free media are cholesterol free.

<p>(A) The mammalian enzyme DHCR24 catalyzes the reduction of a double bond at carbon 24 (arrow) of desmosterol to generate cholesterol. DHCR24^−/− mouse embryonic fibroblasts (MEFs) were isolated and analyzed by PCR genotyping (B) and immunoblotting (C) to confirm the absence of DHCR24. The mutant and wild type alleles are indicated. GAPDH was probed as a loading control. (D) Sterol analysis by high pressure liquid chromatography (HPLC) demonstrated the absence of cholesterol in DHCR24^−/− MEFs once adapted to serum-free media (top panel). Desmosterol replaces cholesterol as the major sterol in these cells. The addition of exogenous cholesterol to DHCR24^−/− MEFs (middle panel) resulted in a sterol profile comparable to DHCR24^+/+ wild type MEFs adapted to serum-free media (lower panel). Commercially available standards were used to determine the retention times of desmosterol and cholesterol.