論文 - 詳細
| RRC ID | 34568 |
|---|---|
| 著者 | Miura H, Inoko H, Tanaka M, Nakaoka H, Kimura M, Gurumurthy CB, Sato M, Ohtsuka M. |
| タイトル | Assessment of Artificial MiRNA Architectures for Higher Knockdown Efficiencies without the Undesired Effects in Mice. |
| ジャーナル | PLoS One |
| Abstract |
RNAi-based strategies have been used for hypomorphic analyses. However, there are technical challenges to achieve robust, reproducible knockdown effect. Here we examined the artificial microRNA (amiRNA) architectures that could provide higher knockdown efficiencies. Using transient and stable transfection assays in cells, we found that simple amiRNA-expression cassettes, that did not contain a marker gene (-MG), displayed higher amiRNA expression and more efficient knockdown than those that contained a marker gene (+MG). Further, we tested this phenomenon in vivo, by analyzing amiRNA-expressing mice that were produced by the pronuclear injection-based targeted transgenesis (PITT) method. While we observed significant silencing of the target gene (eGFP) in +MG hemizygous mice, obtaining -MG amiRNA expression mice, even hemizygotes, was difficult and the animals died perinatally. We obtained only mosaic mice having both "-MG amiRNA" cells and "amiRNA low-expression" cells but they exhibited growth retardation and cataracts, and they could not transmit the -MG amiRNA allele to the next generation. Furthermore, +MG amiRNA homozygotes could not be obtained. These results suggested that excessive amiRNAs transcribed by -MG expression cassettes cause deleterious effects in mice, and the amiRNA expression level in hemizygous +MG amiRNA mice is near the upper limit, where mice can develop normally. In conclusion, the PITT-(+MG amiRNA) system demonstrated here can generate knockdown mouse models that reliably express highest and tolerable levels of amiRNAs. |
| 巻・号 | 10(8) |
| ページ | e0135919 |
| 公開日 | 2015-1-1 |
| DOI | 10.1371/journal.pone.0135919 |
| PII | PONE-D-14-52192 |
| PMID | 26285215 |
| PMC | PMC4540464 |
| MeSH | Animals Cell Line Gene Knockdown Techniques / methods* Gene Silencing Gene Transfer Techniques Genetic Engineering / methods* Green Fluorescent Proteins / genetics Homozygote Male Mice MicroRNAs / genetics* |
| IF | 2.74 |
| 引用数 | 2 |
| WOS 分野 | BIOCHEMISTRY & MOLECULAR BIOLOGY |
| オルトメトリクス指標 |
オルトメトリクス指標項目
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| 最多言及媒体 | Patent(IFI CLAIMS) |
| 各媒体での言及数の合計 | 5 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| 実験動物マウス | RBRC01835 |