Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage phi6 RNA-dependent RNA polymerase

Forskningsoutput: TidskriftsbidragArtikelVetenskapligPeer review

Sammanfattning

The discovery of RNA interference (RNAi) has revolutionized biological research and has a huge potential for therapy. Since small double-stranded RNAs (dsRNAs) are required for various RNAi applications, there is a need for cost-effective methods for producing large quantities of high-quality dsRNA. We present two novel, flexible virus-based systems for the efficient production of dsRNA: (1) an in vitro system utilizing the combination of T7 RNA polymerase and RNA-dependent RNA polymerase (RdRP) of bacteriophage phi 6 to generate dsRNA molecules of practically unlimited length, and (2) an in vivo RNA replication system based on carrier state bacterial cells containing the phi 6 polymerase complex to produce virtually unlimited amounts of dsRNA of up to 4.0 kb. We show that pools of small interfering RNAs (siRNAs) derived from dsRNA produced by these systems significantly decreased the expression of a transgene (eGFP) in HeLa cells and blocked endogenous pro-apoptotic BAX expression and subsequent cell death in cultured sympathetic neurons.
Originalspråkengelska
TidskriftRNA
Volym13
Utgåva3
Sidor (från-till)422-429
Antal sidor8
ISSN1355-8382
DOI
StatusPublicerad - 2007
MoE-publikationstypA1 Tidskriftsartikel-refererad

Vetenskapsgrenar

  • 1183 Växtbiologi, mikrobiologi, virologi
  • 318 Medicinsk bioteknologi

Citera det här

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title = "Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage phi6 RNA-dependent RNA polymerase",
abstract = "The discovery of RNA interference (RNAi) has revolutionized biological research and has a huge potential for therapy. Since small double-stranded RNAs (dsRNAs) are required for various RNAi applications, there is a need for cost-effective methods for producing large quantities of high-quality dsRNA. We present two novel, flexible virus-based systems for the efficient production of dsRNA: (1) an in vitro system utilizing the combination of T7 RNA polymerase and RNA-dependent RNA polymerase (RdRP) of bacteriophage phi 6 to generate dsRNA molecules of practically unlimited length, and (2) an in vivo RNA replication system based on carrier state bacterial cells containing the phi 6 polymerase complex to produce virtually unlimited amounts of dsRNA of up to 4.0 kb. We show that pools of small interfering RNAs (siRNAs) derived from dsRNA produced by these systems significantly decreased the expression of a transgene (eGFP) in HeLa cells and blocked endogenous pro-apoptotic BAX expression and subsequent cell death in cultured sympathetic neurons.",
keywords = "1183 Plant biology, microbiology, virology, 318 Medical biotechnology",
author = "Antti Aalto and Sarin, {Leif Peter} and {Van Dijk}, {Alberdina A} and Mart Saarma and Poranen, {Minna M} and Urmas Arum{\"a}e and Bamford, {Dennis H}",
year = "2007",
doi = "10.1261/rna.348307",
language = "English",
volume = "13",
pages = "422--429",
journal = "RNA",
issn = "1355-8382",
publisher = "Cold Spring Harbor Laboratory in association with the Genetical Society of Great Britain",
number = "3",

}

Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage phi6 RNA-dependent RNA polymerase. / Aalto, Antti; Sarin, Leif Peter; Van Dijk, Alberdina A; Saarma, Mart; Poranen, Minna M; Arumäe, Urmas; Bamford, Dennis H.

I: RNA, Vol. 13, Nr. 3, 2007, s. 422-429.

Forskningsoutput: TidskriftsbidragArtikelVetenskapligPeer review

TY - JOUR

T1 - Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage phi6 RNA-dependent RNA polymerase

AU - Aalto, Antti

AU - Sarin, Leif Peter

AU - Van Dijk, Alberdina A

AU - Saarma, Mart

AU - Poranen, Minna M

AU - Arumäe, Urmas

AU - Bamford, Dennis H

PY - 2007

Y1 - 2007

N2 - The discovery of RNA interference (RNAi) has revolutionized biological research and has a huge potential for therapy. Since small double-stranded RNAs (dsRNAs) are required for various RNAi applications, there is a need for cost-effective methods for producing large quantities of high-quality dsRNA. We present two novel, flexible virus-based systems for the efficient production of dsRNA: (1) an in vitro system utilizing the combination of T7 RNA polymerase and RNA-dependent RNA polymerase (RdRP) of bacteriophage phi 6 to generate dsRNA molecules of practically unlimited length, and (2) an in vivo RNA replication system based on carrier state bacterial cells containing the phi 6 polymerase complex to produce virtually unlimited amounts of dsRNA of up to 4.0 kb. We show that pools of small interfering RNAs (siRNAs) derived from dsRNA produced by these systems significantly decreased the expression of a transgene (eGFP) in HeLa cells and blocked endogenous pro-apoptotic BAX expression and subsequent cell death in cultured sympathetic neurons.

AB - The discovery of RNA interference (RNAi) has revolutionized biological research and has a huge potential for therapy. Since small double-stranded RNAs (dsRNAs) are required for various RNAi applications, there is a need for cost-effective methods for producing large quantities of high-quality dsRNA. We present two novel, flexible virus-based systems for the efficient production of dsRNA: (1) an in vitro system utilizing the combination of T7 RNA polymerase and RNA-dependent RNA polymerase (RdRP) of bacteriophage phi 6 to generate dsRNA molecules of practically unlimited length, and (2) an in vivo RNA replication system based on carrier state bacterial cells containing the phi 6 polymerase complex to produce virtually unlimited amounts of dsRNA of up to 4.0 kb. We show that pools of small interfering RNAs (siRNAs) derived from dsRNA produced by these systems significantly decreased the expression of a transgene (eGFP) in HeLa cells and blocked endogenous pro-apoptotic BAX expression and subsequent cell death in cultured sympathetic neurons.

KW - 1183 Plant biology, microbiology, virology

KW - 318 Medical biotechnology

U2 - 10.1261/rna.348307

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VL - 13

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JO - RNA

JF - RNA

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