From the PNAS web site: pnas.org
The 50 Most-Frequently-Read Contents
in PNAS during February 2004 -- updated monthly
Most-read rankings are recalculated at the beginning of the month.
Rankings are based on hits received by articles archived on this site only.
1.
Sunita Gupta, Rebecca A. Schoer, James E. Egan, Gregory J. Hannon, Vivek Mittal
From the Cover: Inducible, reversible, and stable RNA interference in mammalian cells
PNAS Feb 17, 2004 101: 1927-1932. (In "Genetics")
Abstract
RNA interference is a powerful genetic approach for efficiently silencing target genes. The existing method of gene suppression by the constitutive expression of short hairpin RNAs (shRNAs) allows analysis of the consequences of stably silencing genes but limits the analysis of genes essential for cell survival, cell cycle regulation, and cell development. We have developed an inducible U6 promoter for synthesis of shRNAs in both human and murine cells. Cells containing stably integrated shRNA expression constructs demonstrate stringent dosage- and time-dependent kinetics of induction with undetectable background expression in the absence of the inducer ecdysone. Inducible suppression of human p53 in glioblastoma cells shows striking morphological changes and defects in cell cycle arrest caused by DNA damage, as expected. Remarkably, the inducibility is reversible after withdrawal of the inducer, as observed by reappearance of the protein and a restoration of the original cell phenotype. Inducible and reversible regulation of RNA interference has broad applications in the areas of mammalian genetics and molecular therapeutics.
2.
Peter C. Scacheri, Orit Rozenblatt-Rosen, Natasha J. Caplen, Tyra G. Wolfsberg, Lowell Umayam, Jeffrey C. Lee, Christina M. Hughes, Kalai Selvi Shanmugam, Arindam Bhattacharjee, Matthew Meyerson, Francis S. Collins
Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells
PNAS Feb 17, 2004 101: 1892-1897. (In "Cell_Biology")
Abstract
RNA interference (RNAi) mediated by short interfering RNAs (siRNAs) is a widely used method to analyze gene function. To use RNAi knockdown accurately to infer gene function, it is essential to determine the specificity of siRNA-mediated RNAi. We have assessed the specificity of 10 different siRNAs corresponding to the MEN1 gene by examining the expression of two additional genes, TP53 (p53) and CDKN1A (p21), which are considered functionally unrelated to menin but are sensitive markers of cell state. MEN1 RNA and corresponding protein levels were all reduced after siRNA transfection of HeLa cells, although the degree of inhibition mediated by individual siRNAs varied. Unexpectedly, we observed dramatic and significant changes in protein levels of p53 and p21 that were unrelated to silencing of the target gene. The modulations in p53 and p21 levels were not abolished on titration of the siRNAs, and similar results were obtained in three other cell lines; in none of the cell lines tested did we see an effect on the protein levels of actin. These data suggest that siRNAs can induce nonspecific effects on protein levels that are siRNA sequence dependent but that these effects may be difficult to detect until genes central to a pivotal cellular response, such as p53 and p21, are studied. We find no evidence that activation of the double-stranded RNA-triggered IFN-associated antiviral pathways accounts for these effects, but we speculate that partial complementary sequence matches to off-target genes may result in a micro-RNA-like inhibition of translation. |
