Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells

Paddison, Patrick J.; Caudy, Amy A.; Bernstein, Emily; Hannon, Gregory J.; Conklin, Douglas S.

doi:10.1101/gad.981002

Cited by 1,394 publications

(1,003 citation statements)

References 38 publications

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“…Numerous additional reports indicate that certain individuals or groups are immune to HIV-1 altogether (Baur et al 1989, Bryson et al 1995, Deacon et al 1995, Roques et al 1995, Paxton et al 1996, Kaul et al 2000, Kulkarni et al 2003, while others remain AIDS free for extensive periods of time . In order to develop sufficient human protection against HIV-1 variants, a number of homologous sequences will be required in the endogenous repertoire (Hiroshika et al 2000, Donze & Picard 2002, Hohjoh 2002, Jeong et al 2002, Leirdal & Sioud 2002, Miyagishi & Taira 2002, Paddison et al 2002, Paul et al 2002, Sui et al 2002, Yu et al 2002. Through genetic technology, however, a rapid HIV-1 inhibitory siRNAs specific repertoire can perhaps be designed.…”

Section: Role Of Sirnas In Development Of Hiv-1 Vaccinementioning

confidence: 99%

“…Through genetic technology, however, a rapid HIV-1 inhibitory siRNAs specific repertoire can perhaps be designed. These siRNAs could be encoded and delivered by appropriate vectors and then utilized in preventative or therapeutic vaccines (Hiroshika et al 2000, Donze & Picard 2002, Hohjoh 2002, Jeong et al 2002, Leirdal & Sioud 2002, Miyagishi & Taira 2002, Paddison et al 2002, Paul et al 2002, Sui et al 2002, Yu et al 2002.…”

Section: Role Of Sirnas In Development Of Hiv-1 Vaccinementioning

confidence: 99%

“…One way to answer this question is to evaluate what happens to an organism when certain genes, presumed to be involved in RBGS, are altered. Mutations in several genes render an organism incapable of generating siRNAs; in some cases, these mutations reactivate transposons and retroelements (Ketting et al 1999, Tabara et al 1999, Hirochika et al 2000, Nakayashiki et al 2001, Jeong et al 2002, Paddison et al 2002, Paul et al 2002, Sui et al 2002. Proteins essential for the homologous RNA degradation pathway are highly conserved in fungi, plants, vertebrates, and mammals (reviewed in Grishok & Mello 2002).…”

Section: Issues With Current Models Of Rnaimentioning

confidence: 99%

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RNA interference: The molecular immune system

Bagasra

Prilliman²

2004

Histochem J

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SummaryIntroduction of double-stranded RNA (dsRNA) into cells expressing a homologous gene triggers RNA interference (RNAi), or RNA-based gene silencing (RBGS). The dsRNA degrades corresponding host mRNA into small interfering RNAs (siRNAs) by a protein complex containing Dicer. siRNAs in turn are incorporated into the RNA-induced silencing complex (RISC) that includes helicase, RecA, and exo-and endo-nucleases as well as other proteins. Following its assembly, the RISC guides the RNA degradation machinery to the target RNAs and cleaves the cognate target RNA in a sequence-specific, siRNA-dependent manner. RNAi has now been documented in a wide variety of organisms, including plants, fungi, flies, worms, and more recently, higher mammals. In eukaryotes, dsRNA directed against a range of viruses (i.e., HIV-1, RSV, HPV, poliovirus and others) and endogenous genes can induce sequence-specific inhibition of gene expression. In invertebrates, RNAi can be efficiently triggered by either long dsRNAs or 21-to 23-nt-long siRNAs. However, in jawed vertebrates, dsRNA longer than 30 bp can induce interferon and thus trigger undesirable side effects instead of initiating RNAi. siRNAs have been shown to act as potent inducers of RNAi in cultured mammalian cells. Many investigators have suggested that siRNAs may have evolved as a normal defense against endogenous and exogenous transposons and retroelements. Through a combination of genetic and biochemical approaches, some of the mechanisms underlying RNAi have been described. Recent data in C. elegans shows that two homologs of siRNAs, microRNAs (miRNAs) and tiny noncoding RNAs (tncRNAs) are endogenously expressed. However, many aspects of RNAi-induced gene silencing, including its origins and the selective pressures which maintain it, remain undefined. Its evolutionary history may pass through the more primitive immune functions of prokaryotes involving restriction enzymes that degrade plasmid DNA molecules that enter bacterial cells. RNAi has evolved further among eukaryotes, in which its wide distribution suggests early origins. RNAi seems to be involved in a variety of regulatory and immune functions that may differ among various kingdoms and phyla. We present here proposed mechanisms by which RBGS protects the host against endogenous and exogenous transposons and retroelements. The potential for therapeutic application of RBGS technology in treating viral infections such as HIV is also discussed.

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Section: Role Of Sirnas In Development Of Hiv-1 Vaccinementioning

confidence: 99%

Section: Role Of Sirnas In Development Of Hiv-1 Vaccinementioning

confidence: 99%

Section: Issues With Current Models Of Rnaimentioning

confidence: 99%

See 1 more Smart Citation

RNA interference: The molecular immune system

Bagasra

Prilliman²

2004

Histochem J

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“…Following siRNA sequence selection, a genomic cassette is cloned into appropriate vectors to produce short hairpin RNAs (shRNA), to be processed into siRNA by the cellular machinery. 9,10 Lentivirus-derived vectors enable the high level of stable shRNA essential for gene silencing as a therapeutic strategy for human genetic diseases.…”

Section: Introductionmentioning

confidence: 99%

Allele-specific Col1a1 silencing reduces mutant collagen in fibroblasts from Brtl mouse, a model for classical osteogenesis imperfecta

et al. 2013

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Gene silencing approaches have the potential to become a powerful curative tool for a variety of monogenic diseases caused by gain-of-function mutations. Classical osteogenesis imperfecta (OI), a dominantly inherited bone dysplasia, is characterized in its more severe forms by synthesis of structurally abnormal type I collagen, which exerts a negative effect on extracellular matrix. Specific suppression of the mutant (Mut) allele would convert severe OI forms to the mild type caused by a quantitative defect in normal collagen. Here, we describe the in vitro and ex vivo investigation of a small interfering RNA (siRNA) approach to allele-specific gene silencing using Mut Col1a1 from the Brtl mouse, a well-characterized model for classical human OI. A human embryonic kidney cell line, which expresses the firefly luciferase gene, combined with either wild-type or Mut Brtl Col1a1 exon 23 sequences, was used for the first screening. The siRNAs selected based on their specificity and the corresponding short hairpin RNAs (shRNAs) subcloned in a lentiviral vector were evaluated ex vivo in Brtl fibroblasts for their effect on collagen transcripts and protein. A preferential reduction of the Mut allele of up to 52% was associated with about 40% decrease of the Mut protein, with no alteration of cell proliferation. Interestingly, a downregulation of HSP47, a specific collagen chaperone known to be upregulated in some OI cases, was detected. Our data support further testing of shRNAs and their delivery by lentivirus as a strategy to specifically suppress the Mut allele in mesenchymal stem cells of OI patients for autologous transplantation.

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“…Thus Pol III transcripts normally have 3-4 U residues at the 3 0 end (for a review see Schramm and Hernandez 21 ). A flurry of papers in 2002 from five different groups [22][23][24][25][26] established that short hairpin RNA (shRNA) molecules expressed from U6 or H1 Type 3 Pol III promoters could mediate sequence-specific RNAi-based gene inhibition. All of the groups designed expression units where the RNA sequence expressed from the Pol III promoters contained an inverted repeat of 19-29 nt separated by a short spacer sequence (See Figure 1a).…”

Section: Codon Optimization and Codon Pairing Will Affect Transcriptimentioning

confidence: 99%

Gene Therapy Progress and Prospects: Recent progress in transgene and RNAi expression cassettes

Ill¹,

Chiou²

2005

Gene Ther

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Plasmid expression cassette design must include a thoughtful analysis of potentially every nucleotide comprising a covalently closed circular or end-protected linear DNA. This review will discuss recent studies in unraveling the mechanisms of postdelivery gene silencing, codon optimization and promoter identification. The recent discovery of potent RNA interference (RNAi) mechanisms for sequence-specific gene silencing has also invoked a great deal of interest in development of expression cassettes that can produce double-stranded RNA molecules for RNAi. Expression cassettes based on both RNA polymerase II and polymerase III transcription units that generate double-stranded RNA molecules for RNAi will also be discussed. Gene Therapy (2005) Whether a mammalian cell expression cassette is produced by bacterial amplification, minicircle recombination, or by purely synthetic means, the primary DNA

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Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells

Cited by 1,394 publications

References 38 publications

RNA interference: The molecular immune system

RNA interference: The molecular immune system

Allele-specific Col1a1 silencing reduces mutant collagen in fibroblasts from Brtl mouse, a model for classical osteogenesis imperfecta

Gene Therapy Progress and Prospects: Recent progress in transgene and RNAi expression cassettes

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