[Nonsense RNA: a tool for specifically inhibiting the expression of a gene in vivo].
We describe a general technique to inhibit gene expression in eukaryotic cells. The gene we chose to inhibit was the E. coli LacZ gene (encoding beta-galactosidase), which has previously been cloned into a eukaryotic expression vector [1]. This plasmid is called pCH110. We constructed a variant of pCH110 in which we flipped a 2566 base pair 5' fragment of the LacZ gene into the antiparallel orientation. The plasmid containing this mutated LacZ gene is called pNSLacZ (NS signifies non-sense coding sequence). When equal amounts of pCH110 and pNSLacZ are co-transfected into 3T6 mouse fibroblasts, the beta-galactosidase activity is decreased by approximately a factor of ten. Increasing the ratio of pNSLacZ to pCH110 above 1:1 does not appreciably increase the level of inhibition. Next, we prove the specificity of the inhibition by adding a third gene to the transfection mixture. For this purpose, we used pSVneo beta, a plasmid which expresses a phosphotransferase. We found that even when the beta-galactosidase activity was diminished by a factor of 10, the phosphotransferase activity was unaffected. Therefore, we have demonstrated that: the presence of an antiparallel copy of the LacZ gene results in a significant and specific diminution of the LacZ gene's expression; only a fraction of the LacZ gene needs to be in the antiparallel orientation in order to observe this effect. These results suggest that this technique can serve as a tool to decrease the level of gene expression in order to study the function of specific genes, or as a therapeutic manoeuvre in the treatment of disorders of abnormal gene expression.
D000818:Animals; D002460:Cell Line; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005696:Galactosidases / Q000235:genetics*; D005786:Gene Expression Regulation; D051379:Mice; D010957:Plasmids; D012313:RNA / Q000378:metabolism*; D013539:Simian virus 40 / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
D012313:RNA; D005696:Galactosidases; D001616:beta-Galactosidase
L'ARN non sens (nsARN): un outil pour inactiver spécifiquement l'expression d'un gène donné in vivo.