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Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord
Published on: March 18, 2013
Constitutive and conditional RNAi transgenesis in mice
Aljoscha Kleinhammer1, Wolfgang Wurst, Ralf Kühn
1Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Developmental Genetics, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany. aljoscha.kleinhammer@helmholtz-muenchen.de
Methods (San Diego, Calif.)
|December 28, 2010
Summary
This study presents three strategies for controlling gene silencing in mice using short hairpin (sh) RNA. These methods enable efficient gene knockdown for functional genomic analysis in vivo.
Area of Science:
- Genetics
- Molecular Biology
- Genomics
Background:
- RNA interference (RNAi) is a key method for understanding mammalian gene function.
- Short hairpin (sh) RNAs offer a rapid alternative to traditional knockout methods for gene silencing.
- Existing methods for in vivo gene knockdown require optimization for specific applications.
Purpose of the Study:
- To develop and present three distinct strategies for controlled gene silencing in mice using shRNA.
- To enable both germline and inducible, cell type-specific gene knockdown.
- To provide a versatile platform for in vivo functional genomic studies.
Main Methods:
- Utilizing shRNAs expressed from stably integrated vectors for RNAi.
- Implementing Cre/loxP for permanent gene silencing and tetO/tTA for reversible silencing.
- Employing recombinase-mediated cassette exchange (RMCE) to insert shRNAs into the Rosa26 locus in ES cells.
- Generating shRNA transgenic mice from engineered ES cells.
Main Results:
- Demonstrated three effective strategies for controlled gene silencing in mice.
- Achieved efficient gene knockdown through both permanent and reversible systems.
- Successfully generated shRNA transgenic mice using RMCE in ES cells.
Conclusions:
- The described strategies offer robust control over gene silencing in mice.
- This approach facilitates rapid in vivo functional analysis through efficient gene knockdown.
- The Rosa26 docking site and RMCE provide a reliable platform for shRNA integration and mouse generation.
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To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
02:15Experimental RNAi
RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...