Long endogenous dsRNAs can induce complete gene silencing in mammalian cells and primary cultures.
Recently, double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) has rapidly developed to a powerful instrument for specific silencing of gene expression in several organisms, including Caenorhabditis elegans, Drosophila melanogaster, and plants. The finding that synthetic small interfering RNAs (siRNAs) of 21 nt as well as stable, endogenously expressed, large dsRNA are suited to specifically induce gene silencing in mammalian cells offered the possibility of expanding this technique to mammalian systems. In this work, we engineered stably transfected human cells that express large dsRNAs mediating specific posttranscriptional silencing of genes. We used this technique to specifically silence genes coding for glucosylceramide synthase (GCS), the sphingolipid activator protein precursor (SAP), and glucocerebrosidase (GBA), all implicated in glycosphingolipid metabolism. From a 1600-bp inverted repeat DNA template, a dsRNA of 800 bp is expressed and predicted to mediate the specific suppression of the corresponding gene by RNAi. Remarkably, we were able to use this method to achieve complete inhibition of those genes we targeted in different cultured human cell lists. These findings testify to the generality of RNAi application in suppressing gene expression in mammalian cells.
Diallo
M
M
Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Germany.
Arenz
C
C
Schmitz
K
K
Sandhoff
K
K
Schepers
U
U
D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003001:Cloning, Molecular; D020868:Gene Silencing / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008322:Mammals; D008546:Melanoma, Experimental; D051379:Mice; D012330:RNA, Double-Stranded / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D015183:Restriction Mapping; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering