Showing papers by "Makoto Miyagishi published in 2002"

U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells

Abstract: The first evidence for gene disruption by double-stranded RNA (dsRNA) came from careful analysis in Caenorhabditis elegans. This phenomenon, called RNA interference (RNAi), was observed subsequently in various organisms, including plants, nematodes, Drosophila, and protozoans. Very recently, it has been reported that in mammalian cells, 21- or 22-nucleotide (nt) RNAs with 2-nt 3' overhangs (small inhibitory RNAs, siRNAs) exhibit an RNAi effect. This is because siRNAs are not recognized by the well-characterized host defense system against viral infections, involving dsRNA-dependent inhibition of protein synthesis. However, the current method for introducing synthetic siRNA into cells by lipofection restricts the range of applications of RNAi as a result of the low transfection efficiencies in some cell types and/or short-term persistence of silencing effects. Here, we report a vector-based siRNA expression system that can induce RNAi in mammalian cells. This technical advance for silencing gene expression not only facilitates a wide range of functional analysis of mammalian genes but might also allow therapeutic applications by means of vector-mediated RNAi.

Effects on RNA interference in gene expression (RNAi) in cultured mammalian cells of mismatches and the introduction of chemical modifications at the 3'-ends of siRNAs.

Abstract: The highly specific posttranscriptional silencing of gene expression induced by double-stranded RNA (dsRNA) is known as RNA interference (RNAi) and has been demonstrated in plants, nematodes, Drosophila, and protozoa, as well as in mammalian cells. The suppression of expression of specific genes by chemically synthesized 21-nucleotide (21-nt) RNA duplexes has been achieved in various lines of mammalian cells, and this technique might prove to be a valuable tool in efforts to analyze biologic functions of genes in mammalian cells. In order to investigate the utility of potential modifications that can be introduced into small interfering RNAs (siRNAs) and also to study their functional anatomy, we synthesized different types of siRNA targeted to mRNA of Jun dimerization protein 2 (JDP2). Our detailed analysis demonstrated that siRNAs with only one mismatch, relative to the target, on the antisense strand had reduced RNAi effect, whereas the corresponding mutation on the sense strand did not interfere with the RNAi. Moreover, one 2-hydroxyethylphosphate (hp) substitution at the 3'-end of the antisense strand but not of the sense strand also prevented RNAi, whereas a related modification at the 3'-end of either strand, using 2'-O,4'-C-ethylene thymidine (eT), which is a component of ethylene-bridge nucleic acids (ENA), completely abolished RNAi. These results support the hypothesis that the two strands have different functions in RNAi in cultured mammalian cells and indicate that their chemical modification of siRNAs at the 3'-end of the sense strand exclusively is possible, without loss of RNAi activity, depending on the type of modification. Because modification at the 3'-end of the antisense strand by hp or eT abolished the RNAi effect, it appears possible that the 3'-end is recognized by the RNA-induced silencing complex (RISC).

Induction of apoptosis in HeLa cells with siRNA expression vector targeted against bcl-2.

Abstract: RNA interference (RNAi) is the process by which double-stranded RNA (dsRNA) directs sequence-specific gene silencing in animal and plant cells. In mammalian cells, 21- or 22-nucleotide (nt) RNAs with 2-nt 3' overhangs small inhibitory RNAs (siRNAs) exhibit an RNAi effect. Very recently, we and others have developed a vector-based siRNA expression system that can induce RNAi in mammalian cells. In this report, to apply this system to oncogene therapy, we tried to suppress the expression of the bcl-2 gene, which is known as a key molecule in the regulation of apoptosis or programmed cell death, by using the siRNA expression system. Western blotting analysis revealed that this siRNA expression vector against bcl-2 suppressed the expression of the bcl-2 protein. Furthermore, HeLa cells which were transiently transfected with the siRNA expression vector against bcl-2 and were subsequently treated with doxorubicin efficiently underwent apoptosis, concomitant with the repression of the bcl-2 gene. These results demonstrate that the siRNA expression vector against bcl-2 has a potential as therapeutic agent for a variety of cancers by down-regulating bcl-2 gene expression.

siRNA expression system and method for producing functional gene knock-down cell using the system

Abstract: An in vivo siRNA expression system whereby an si(small interfering)RNA is expressed in cells. This system has an antisense code DNA encoding an antisense RNA to a domain of a target gene mRNA, a sense code DNA encoding a sense RNA in a domain of the target gene mRNA, and one or more promoters for expressing the above-described antisense RNA and the above-described sense RNA by the above-described antisense code DNA and the above-described sense code DNA.

Sirna expression system and process for producing functional gene knockdown cell or the like using the same

Abstract: An in vivo siRNA expression system for expressing si (small-interfering) RNA in cells which is provided with an antisense code DNA encoding an antisense RNA to any domain in a target gene mRNA, a sense code DNA encoding a sense RNA of any domain of the target gene mRNA, and one or more promoters enabling the expression of the antisense RNA and the sense RNA from the antisense code DNA and the sense code DNA.

Development and application of siRNA expression vector

Abstract: RNA interference (RNAi) is a sequence-specific silencing phenomenon, which is induced by double-stranded RNA (dsRNA) and mediated through an evolutionarily conserved mechanism from plants to mammals. In mammalian cells, it has recently been reported that 21- or 22-nucleotide (nt) RNAs with 2-nt 3' overhangs (siRNA) induce RNAi without induction of the dsRNA-dependent inhibition of protein synthesis, known as the host defense system against viral infections. Moreover, we and other have developed siRNA expression systems utilizing a pol III promoter. Here we report a comparative analysis among various siRNA expression vectors and also demonstrate a regulatable RNAi in cells by using a tetracycline-controlled U6 promoter.

Systeme d'expression d'arnsi et procede de production de cellule knockdown a gene fonctionnel ou analogue utilisant ce systeme

Abstract: L'invention concerne un systeme d'expression d'ARNsi in vivo permettant d'exprimer de l'ARNsi (petits ARN d'interference) dans des cellules. Ce systeme comporte un ADN code antisens qui code pour un ARN antisens lie a un domaine d'ARNm de gene cible ; un ADN code sens codant pour un ARN sens dans un domaine de l'ARNm du gene cible ; et un ou plusieurs promoteurs pour exprimer l'ARN antisens et l'ARN sens decrits au moyen de l'ADN code antisens et de l'ADN code sens decrits.

Systeme d'expression d'arn si et procede de production de cellules d'inactivation de genes fonctionnelles et analogues au moyen de ce systeme

Abstract: L'invention concerne un systeme d'expression d'ARN si in vivo pour l'expression d'ARN si (a faible interference) dans des cellules comportant un ADN a code anti-sens, codant un ARN anti-sens dans tout domaine dans un gene cible ARN m, un ADN a code sens, codant un ARN de tout domaine de ARN m de gene cible, et un ou plusieurs promoteurs permettant l'expression de ARN anti-sens et de ARN sens a partir de ADN a code anti-sens et de ADN a code sens.