Subcellular Distribution of Small Interfering RNA: Directed Delivery Through RNA Polymerase III Expression Cassettes and Localization by In Situ Hybridization

Paul, Cynthia P.

doi:10.1016/s0076-6879(04)92008-3

Cited by 6 publications

(4 citation statements)

References 42 publications

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“…However, an alternative method to express short RNA molecules may be required for particular experiments. A need to screen or modify Pol III promoters in order to obtain a strong and reliable inhibition in a particular cell type or organ has been reported (34,35). Occasionally, irreversible silencing of Pol III-dependent transcription was observed in stable expression systems.…”

Section: Discussionmentioning

confidence: 99%

Development of a species-specific RNA polymerase I-based shRNA expression vector

Verca

Weber

Mayer

et al. 2006

Nucleic Acids Research

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RNA interference (RNAi) can be induced in vitro either by application of synthetic short interfering RNAs (siRNAs), or by intracellular expression of siRNAs or short hairpin RNAs (shRNAs) from transfected vectors. The most widely used promoters for siRNA/shRNA expression are based on polymerase III (Pol III)-dependent transcription. We developed an alternative vector for siRNA/shRNA expression, using a mouse RNA polymerase I (Pol I) promoter. Pol I-dependent transcription serves in cells for production of ribosomal RNA (rRNA), and as such, is ubiquitously and stably active in different cell types. As Pol I-dependent transcription is highly species-specific, Pol I-based system provides an important biosafety advantage with respect to silencing of genes with unknown functions.

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Section: Discussionmentioning

confidence: 99%

Development of a species-specific RNA polymerase I-based shRNA expression vector

Verca

Weber

Mayer

et al. 2006

Nucleic Acids Research

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“…Vector-based systems using RNA polymerase III promoters that stably produce siRNA from short hairpin RNA (shRNA) molecules have been established [1]–[5]. The production of RNAi-mediated gene knockdown transgenic mice (transgenic RNAi mice) has been demonstrated with GFP transgenic mice, which introduced a shRNA expression vector against GFP mRNA (pGtoR) [6], [7].…”

Section: Introductionmentioning

confidence: 99%

Multiple Renal Cyst Development but Not Situs Abnormalities in Transgenic RNAi Mice against Inv::GFP Rescue Gene

et al. 2014

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In this study we generated RNA interference (RNAi)-mediated gene knockdown transgenic mice (transgenic RNAi mice) against the functional Inv gene. Inv mutant mice show consistently reversed internal organs (situs inversus), multiple renal cysts and neonatal lethality. The Inv::GFP-rescue mice, which introduced the Inv::GFP fusion gene, can rescue inv mutant mice phenotypes. This indicates that the Inv::GFP gene is functional in vivo. To analyze the physiological functions of the Inv gene, and to demonstrate the availability of transgenic RNAi mice, we introduced a short hairpin RNA expression vector against GFP mRNA into Inv::GFP-rescue mice and analyzed the gene silencing effects and Inv functions by examining phenotypes. Transgenic RNAi mice with the Inv::GFP-rescue gene (Inv-KD mice) down-regulated Inv::GFP fusion protein and showed hypomorphic phenotypes of inv mutant mice, such as renal cyst development, but not situs abnormalities or postnatal lethality. This indicates that shRNAi-mediated gene silencing systems that target the tag sequence of the fusion gene work properly in vivo, and suggests that a relatively high level of Inv protein is required for kidney development in contrast to left/right axis determination. Inv::GFP protein was significantly down-regulated in the germ cells of Inv-KD mice testis compared with somatic cells, suggesting the existence of a testicular germ cell-specific enhanced RNAi system that regulates germ cell development. The Inv-KD mouse is useful for studying Inv gene functions in adult tissue that are unable to be analyzed in inv mutant mice showing postnatal lethality. In addition, the shRNA-based gene silencing system against the tag sequence of the fusion gene can be utilized as a new technique to regulate gene expression in either in vitro or in vivo experiments.

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“…One method uses modified nucleic acid probes to increase affinity and specificity; such probes include radioactively labeled synthetic RNA oligonucleotide probes, 2 0 -O-methyl RNA oligonucleotides, and locked nucleic acids (LNA). [36][37][38] The most successful method so far uses LNA probes, in which the ribose of the nucleotide contains a bridge connecting its 2 0 oxygen to its 4 0 carbon that locks the ribose in the 3 0 -endo conformation, thereby increasing base stacking forces and backbone preorganization. 37,39 This modification significantly increases the specificity and sensitivity of binding to mature miRNAs.…”

Section: Introductionmentioning

confidence: 99%

Observation ofmiRNAGene Expression in Zebrafish Embryos byIn SituHybridization to MicroRNA Primary Transcripts

Yan

DeLaurier

et al. 2011

Zebrafish

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MicroRNAs (miRNAs) add a previously unexpected layer to the post-transcriptional regulation of protein production. Although locked nucleic acids (LNAs) reveal the distribution of mature miRNAs by in situ hybridization (ISH) experiments in zebrafish and other organisms, high cost has restricted their use. Further, LNA probes designed to recognize mature miRNAs do not distinguish expression patterns of two miRNA genes that produce the same mature miRNA sequence. Riboprobes are substantially less expensive than LNAs, but have not been used to detect miRNA gene expression because they do not bind with high affinity to the short, 22-nucleotide-long mature miRNAs. To solve these problems, we capitalized on the fact that miRNAs are initially transcribed into long primary transcripts (pri-mRNAs). We show here that conventional digoxigenin-labeled riboprobes can bind to primary miRNA transcripts in zebrafish embryos. We tested intergenic and intronic miRNAs (miR-10d, miR-21, miR-27a, miR-126a, miR-126b, miR-138, miR-140, miR-144, miR-196a1, miR-196a2, miR-196a2b [miR-196c], miR-196b, miR-196b1b [miR-196d], miR-199, miR-214, miR-200, and miR-222) in whole mounts and some of these in histological sections. Results showed that pri-miRNA ISH provides an attractive and cost-effective tool to study miRNA expression by ISH. We use this method to show that miR-126a and miR126b are transcribed in the caudal vasculature in the pattern of their neighboring gene ci116 or host gene egfl7, respectively, and that the chondrocyte miRNA mir-140 lies downstream of Sox9 in development of the craniofacial skeleton.

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Subcellular Distribution of Small Interfering RNA: Directed Delivery Through RNA Polymerase III Expression Cassettes and Localization by In Situ Hybridization

Cited by 6 publications

References 42 publications

Development of a species-specific RNA polymerase I-based shRNA expression vector

Development of a species-specific RNA polymerase I-based shRNA expression vector

Multiple Renal Cyst Development but Not Situs Abnormalities in Transgenic RNAi Mice against Inv::GFP Rescue Gene

Observation ofmiRNAGene Expression in Zebrafish Embryos byIn SituHybridization to MicroRNA Primary Transcripts

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