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Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction
Published on: August 25, 2017
Introduction of tau mutation into cultured Rat1-R12 cells by gene targeting, using recombinant adeno-associated virus
Hiroko Shimada1, Kahori Numazawa, Tsukasa Sasaki
1Department of Sleep Disorder Research (Alfressa), Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
Abstract:
We aim to develop a cultured cell model, to serve as a system with which the altered circadian phenotypes produced by the clock gene variations could be studied in vitro. Tau mutation, which shortens the circadian period of hamsters and mice, was introduced into the CK1epsilon locus of cultured Rat1-R12 cells by gene targeting mediated by a recombinant adeno-associated virus (rAAV) vector. After transduction of Rat1-R12 cells with rAAV, about 0.14% of the drug-resistant cells underwent gene targeting at CK1epsilon locus. Of the three clones isolated, only one carried the targeted allele of tau mutation and two carried the targeted wild-type allele. The clone with the targeted tau mutant allele exhibited a significantly shorter circadian period compared to the clone with targeted wild-type allele. rAAV-mediated gene targeting in cultured somatic cells is a convenient and powerful tool for analyzing the phenotypic outcome of clock gene variations, and for elucidating the pathogenesis of the disorders associated with abnormal circadian rhythmicity.
Insights
Researchers created a cell model to study circadian rhythm disorders. Introducing a tau mutation into Rat1-R12 cells via recombinant adeno-associated virus (rAAV) resulted in a shorter circadian period, validating this method.
Area of Science:
- Chronobiology
- Molecular Biology
- Cell Biology
Background:
- Circadian rhythm disorders are linked to clock gene variations.
- In vitro models are needed to study these genetic alterations and their phenotypic outcomes.
- The tau mutation is known to shorten circadian periods in vivo.
Purpose of the Study:
- To develop a cultured cell model for studying circadian phenotypes caused by clock gene variations.
- To investigate the effects of the tau mutation in a cellular system.
- To establish a method for analyzing clock gene function in vitro.
Main Methods:
- Gene targeting using a recombinant adeno-associated virus (rAAV) vector.
- Introduction of the tau mutation into the CK1epsilon locus of Rat1-R12 cells.
- Isolation and characterization of drug-resistant cell clones.
Main Results:
- Successful gene targeting at the CK1epsilon locus was achieved in approximately 0.14% of drug-resistant cells.
- One clone carried the targeted tau mutant allele, while two carried the targeted wild-type allele.
- The clone with the tau mutant allele displayed a significantly shorter circadian period compared to the wild-type clone.
Conclusions:
- Recombinant adeno-associated virus (rAAV)-mediated gene targeting is an effective tool for creating cellular models of circadian gene variations.
- This in vitro system allows for the analysis of phenotypic consequences of clock gene mutations.
- The developed model aids in understanding the pathogenesis of circadian rhythm disorders.
