Constitutive and conditional RNAi transgenesis in mice
| Autor: | Aljoscha Kleinhammer, Wolfgang Wurst, Ralf Kühn |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
RNA
Untranslated Transcription Genetic metabolism [Recombinases] Transgene Genetic Vectors Cre recombinase Mice Transgenic Biology Transfection General Biochemistry Genetics and Molecular Biology Small hairpin RNA Recombinases Mice RNA interference metabolism [Embryonic Stem Cells] pharmacology [Doxycycline] Gt(ROSA)26Sor non-coding RNA mouse Gene silencing Animals Humans RNA Small Interfering Molecular Biology Embryonic Stem Cells Regulation of gene expression Gene knockdown Recombinase-mediated cassette exchange Proteins genetics [Proteins] Molecular biology Phenotype Gene Expression Regulation Genes biosynthesis [RNA Small Interfering] Doxycycline ddc:540 methods [Transfection] RNA Interference Genetic Engineering |
| Zdroj: | Methods 53(4), 430-436 (2011). doi:10.1016/j.ymeth.2010.12.015 |
| ISSN: | 1095-9130 |
| DOI: | 10.1016/j.ymeth.2010.12.015 |
| Popis: | Gene silencing by RNA interference (RNAi) has become a routine method for extracting function from the mammalian genome. Short hairpin (sh) RNAs expressed from stably integrated vectors mediate RNAi both in cultured cells and mice and present therefore a fast alternative to conventional knockout approaches. We describe three strategies to control gene silencing in mice by shRNA expression that can be applied to any transcript of interest. The strategies include germline and inducible cell type-specific knockdowns, which depending on the molecular switch applied can be either permanent (Cre/loxP) or reversible (tetO/tTA). For reliable expression the shRNAs of interest are knocked into a pre-engineered Rosa26 docking site by recombinase mediated cassette exchange (RMCE). ES cells expressing the shRNA of interest can then be used to generate shRNA transgenic mice. The high efficiency of RMCE in ES cells enables the fast production of knockdown mice for in vivo functional analysis. |
| Databáze: | OpenAIRE |
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