The enzyme DHCR24 (24-dehydrocholesterol reductase) functions in the Bloch pathway of cholesterol biosynthesis and is capable of reducing several sterol intermediates. It catalyzes the reduction of the double bond at the C24 position present in all intermediates, resulting in the formation of more saturated sterols, including cholesterol as the final product. Impaired DHCR24 activity leads to the accumulation of sterol intermediates, which can affect various cellular processes. The aim of this Undergraduate thesis was to establish an inducible lentiviral shRNA system for specific and temporally controlled silencing of DHCR24 in hepatic cell lines and to quantitatively determine changes in the sterol profile using LC-MS/MS. A shRNA construct targeting DHCR24 was introduced into three hepatic cell lines (HepG2, Huh7, and Snu449) using lentiviral particles. Following genomic integration, the shRNA cassette is stably maintained through subsequent cell divisions. The inducible shRNA system, regulated by an IPTG-inducible promoter, enables controlled activation or suppression of gene silencing. This is particularly important for DHCR24, as its complete loss impairs cell growth and survival. Following sterol extraction and quantification by LC-MS/MS, successful DHCR24 silencing was demonstrated in the Snu449 MOI5 cell line that had not been subjected to antibiotic selection.
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