Functional analysis of cholesterol biosynthesis by RNA interference.

[child syndrome]

Inborn errors of cholesterol biosynthesis caused by dysfunctionality of single enzymes are known to cause severe malformation syndromes like X-linked chondrodysplasia punctata (CDPX 2 ), CHILD syndrome or Smith-Lemli-Opitz-syndrome (SLOS ). In this study we established the method of RNA interference (RNAi ) for analyzing the molecular mechanisms underlying disrupted cholesterol biosynthesis . For different genes involved in the cholesterol biosynthesis pathway-NAD (P ) dependent steroid dehydrogenase-like (NSDHL ), 17 -beta hydroxysteroid dehydrogenase type 7 (HSD 17 B 7 ) and emopamil binding protein (EBP )-shRNA sequences were designed and tested for their effectiveness . For a better comparability of the experiments and to avoid different transfection efficiencies , examined shRNA sequences which reached a knock down of at least 80 % were stably transfected in a HeLa cell line with a tetracycline-regulated expression (HeLa T -REx ). These stable transfected cell lines represent novel tools for the analysis of cholesterol biosynthesis .