Functional analysis of cholesterol biosynthesis by RNA interference.
Inborn errors of cholesterol biosynthesis caused by dysfunctionality of single enzymes are known to cause severe malformation syndromes like X-linked chondrodysplasia punctata (CDPX2), 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 (HSD17B7) 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.
Guggenberger
Christina
C
GSF-National Research Center for Environment and Health, Institute of Experimental Genetics, Genome Analysis Center, Ingolstaedter Landstr. 1, 85764 Neuherberg, Germany.
Ilgen
Denise
D
Adamski
Jerzy
J
D015096:3-Hydroxysteroid Dehydrogenases / Q000235:genetics; D001483:Base Sequence; D002784:Cholesterol / Q000096:biosynthesis*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D013251:Steroid Isomerases / Q000235:genetics; D014162:Transfection / Q000379:methods
D047908:Intracellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; C116431:src kinase associated phosphoprotein 2; D002784:Cholesterol; D015096:3-Hydroxysteroid Dehydrogenases; C478776:Nsdhl protein, human; D013251:Steroid Isomerases; C090445:EBP protein, human