Targeting genes in living mammals by RNA interference

Podolská, Kateřina; Svoboda, Petr

doi:10.1093/bfgp/elr013

Cited by 12 publications

(13 citation statements)

References 89 publications

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“…The constitutive expression of certain amiRNA sequences that leads to embryonic lethality can eventually result in unavailability of a model for further studies 24 25 . A conditional expression strategy offers the best solution in such cases to generate a viable model.…”

Section: Resultsmentioning

confidence: 99%

“…To overcome the limitations associated with constitutive knockdown, such as lack of tissue-specificity or lethality due to RNAi, conditional approaches using Cre- lox P or inducible RNAi using tetracycline have been utilized 24 25 . In a Cre- lox P-based conditional RNAi approach, to prevent generation of constitutively active shRNA, lox P-flanked stuffer sequences were inserted into the shRNA loop or between the promoter and the shRNA 24 25 . In this case, RNAi is induced after Cre-mediated excision of stuffer sequence from the cassette.…”

Section: Discussionmentioning

confidence: 99%

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CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA

Miura

Gurumurthy

Sato

et al. 2015

Sci Rep

126

148

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Knockdown mouse models, where gene dosages can be modulated, provide valuable insights into gene function. Typically, such models are generated by embryonic stem (ES) cell-based targeted insertion, or pronuclear injection, of the knockdown expression cassette. However, these methods are associated with laborious and time-consuming steps, such as the generation of large constructs with elements needed for expression of a functional RNAi-cassette, ES-cell handling, or screening for mice with the desired knockdown effect. Here, we demonstrate that reliable knockdown models can be generated by targeted insertion of artificial microRNA (amiRNA) sequences into a specific locus in the genome [such as intronic regions of endogenous eukaryotic translation elongation factor 2 (eEF-2) gene] using the Clustered Regularly Interspaced Short Palindromic Repeats/Crispr associated 9 (CRISPR/Cas9) system. We used in vitro synthesized single-stranded DNAs (about 0.5-kb long) that code for amiRNA sequences as repair templates in CRISPR/Cas9 mutagenesis. Using this approach we demonstrate that amiRNA cassettes against exogenous (eGFP) or endogenous [orthodenticle homeobox 2 (Otx2)] genes can be efficiently targeted to a predetermined locus in the genome and result in knockdown of gene expression. We also provide a strategy to establish conditional knockdown models with this method.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA

Miura

Gurumurthy

Sato

et al. 2015

Sci Rep

126

148

View full text Add to dashboard Cite

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“…siRNAs, approximately 21-nucleotide long, are produced in the cell from longer double-stranded RNAs or can be synthesised from small hairpin RNAs (shRNAs) transcribed from genetic constructs produced in the laboratory and introduced into the cells. RNAi has been used for the functional knockdown of specific proteins in several experimental systems, from cultured cells [1] to complete organisms, including mammals [2]. Typically, knockdown transgenic animals are produced by the introduction of the RNAi construct in the cells by lentiviral vectors or by pronuclear microinjection of one-cell embryos.…”

Section: Introductionmentioning

confidence: 99%

“…Typically, knockdown transgenic animals are produced by the introduction of the RNAi construct in the cells by lentiviral vectors or by pronuclear microinjection of one-cell embryos. It is also possible to generate knockdown animals from genetically modified ES cells [3]; for a review about the application of this technology to living mammals, see [2]. …”

Section: Introductionmentioning

confidence: 99%

RNAi-Mediated Knockdown of IKK1 in Transgenic Mice Using a Transgenic Construct Containing the Human H1 Promoter

Moreno-Maldonado

Murillas

Navarro

et al. 2014

The Scientific World Journal

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Inhibition of gene expression through siRNAs is a tool increasingly used for the study of gene function in model systems, including transgenic mice. To achieve perdurable effects, the stable expression of siRNAs by an integrated transgenic construct is necessary. For transgenic siRNA expression, promoters transcribed by either RNApol II or III (such as U6 or H1 promoters) can be used. Relatively large amounts of small RNAs synthesis are achieved when using RNApol III promoters, which can be advantageous in knockdown experiments. To study the feasibility of H1 promoter-driven RNAi-expressing constructs for protein knockdown in transgenic mice, we chose IKK1 as the target gene. Our results indicate that constructs containing the H1 promoter are sensitive to the presence of prokaryotic sequences and to transgene position effects, similar to RNApol II promoters-driven constructs. We observed variable expression levels of transgenic siRNA among different tissues and animals and a reduction of up to 80% in IKK1 expression. Furthermore, IKK1 knockdown led to hair follicle alterations. In summary, we show that constructs directed by the H1 promoter can be used for knockdown of genes of interest in different organs and for the generation of animal models complementary to knockout and overexpression models.

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“…The use of RNA interference (RNAi) technology has proven to be a very useful tool in disease modelling in mammalian systems [5]. The use of different promoters has allowed for tissue-specific expression of miRNAs, therefore restricting gene knockdown to targeted areas/organs, and also conditional knockdown with the use of Tet-on promoters (the Cre/loxP system is used in mice) [6].…”

Section: Introductionmentioning

confidence: 99%

In VivoTesting of MicroRNA-Mediated Gene Knockdown in Zebrafish

Leong

Lan

Skinner

et al. 2012

Journal of Biomedicine and Biotechnology

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The zebrafish (Danio rerio) has become an attractive model for human disease modeling as there are a large number of orthologous genes that encode similar proteins to those found in humans. The number of tools available to manipulate the zebrafish genome is limited and many currently used techniques are only effective during early development (such as morpholino-based antisense technology) or it is phenotypically driven and does not offer targeted gene knockdown (such as chemical mutagenesis). The use of RNA interference has been met with controversy as off-target effects can make interpreting phenotypic outcomes difficult; however, this has been resolved by creating zebrafish lines that contain stably integrated miRNA constructs that target the desired gene of interest. In this study, we show that a commercially available miRNA vector system with a mouse-derived miRNA backbone is functional in zebrafish and is effective in causing eGFP knockdown in a transient in vivo eGFP sensor assay system. We chose to apply this system to the knockdown of transcripts that are implicated in the human cardiac disorder, Long QT syndrome.

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Targeting genes in living mammals by RNA interference

Cited by 12 publications

References 89 publications

CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA

CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA

RNAi-Mediated Knockdown of IKK1 in Transgenic Mice Using a Transgenic Construct Containing the Human H1 Promoter

In VivoTesting of MicroRNA-Mediated Gene Knockdown in Zebrafish

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