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 CK1ε 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 CK1ε 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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Balsalobre A, Damiola F, Schibler U (1998) A serum shock induces circadian gene expression in mammalian tissue culture cells. Cell 93:929–937. doi:10.1016/S0092-8674(00)81199-X
Brown SA, Fleury-Olela F, Nagoshi E et al (2005) The period length of fibroblast circadian gene expression varies widely among human individuals. PLoS Biol 3:1813–1818. doi:10.1371/journal.pbio.0030338
Davies IR, Mason R (1994) Tau-mutant hamster SCN clock neurones express a 20 h firing rate rhythm in vitro. Neuroreport 5:2165–2168. doi:10.1097/00001756-199410270-00044
Dong JY, Fan PD, Frizzell RA (1996) Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum Gene Ther 7:2101–2112. doi:10.1089/hum.1996.7.17-2101
Ebisawa T, Uchiyama M, Kajimura N et al (2001) Association of structural polymorphisms in the human period3 gene with delayed sleep phase syndrome. EMBO Rep 2:342–346. doi:10.1093/embo-reports/kve070
Gachon F, Nagoshi E, Brown SA et al (2004) The mammalian circadian timing system: from gene expression to physiology. Chromosoma 113:103–112. doi:10.1007/s00412-004-0296-2
Johansson C, Willeit M, Smedh C et al (2003) Circadian clock-related polymorphisms in seasonal affective disorder and their relevance to diurnal preference. Neuropsychopharmacology 28:734–739. doi:10.1038/sj.npp.1300121
Kanegae Y, Takamori K, Sato Y et al (1996) Efficient gene activation system on mammalian cell chromosomes using recombinant adenovirus producing Cre recombinase. Gene 181:207–212. doi:10.1016/S0378-1119(96)00516-1
Kohli M, Rago C, Lengauer C et al (2004) Facile methods for generating human somatic cell gene knockouts using recombinant adeno-associated viruses. Nucleic Acids Res 32:e3. doi:10.1093/nar/gnh009
Liu AC, Welsh DK, Ko CH et al (2007) Intercellular coupling confers robustness against mutations in the SCN circadian clock network. Cell 129:605–616. doi:10.1016/j.cell.2007.02.047
Lowrey PL, Shimomura K, Antoch MP et al (2000) Positional syntenic cloning and functional characterization of the mammalian circadian mutation tau. Science 288:483–492. doi:10.1126/science.288.5465.483
McClung CA (2007) Circadian genes, rhythms and the biology of mood disorders. Pharmacol Ther 114:222–232. doi:10.1016/j.pharmthera.2007.02.003
Meng QJ, Logunova L, Maywood ES et al (2008) Setting clock speed in mammals: the CK1 epsilon tau mutation in mice accelerates circadian pacemakers by selectively destabilizing PERIOD proteins. Neuron 58:78–88. doi:10.1016/j.neuron.2008.01.019
Nagoshi E, Saini C, Bauer C et al (2004) Circadian gene expression in individual fibroblasts: cell-autonomous and self-sustained oscillators pass time to daughter cells. Cell 119:693–705
Ptácek LJ, Jones CR, Fu YH (2007) Novel insights from genetic and molecular characterization of the human clock. Cold Spring Harb Symp Quant Biol 72:273–277. doi:10.1101/sqb.2007.72.017
Russell DW, Hirata RK (1998) Human gene targeting by viral vectors. Nat Genet 18:325–330. doi:10.1038/ng0498-325
Takano A, Uchiyama M, Kajimura N et al (2004) A missense variation in human casein kinase I epsilon gene that induces functional alteration and shows an inverse association with circadian rhythm sleep disorders. Neuropsychopharmacology 29:1901–1909. doi:10.1038/sj.npp.1300503
Vasileva A, Jessberger R (2005) Precise hit: adeno-associated virus in gene targeting. Nat Rev Microbiol 3:837–847. doi:10.1038/nrmicro1266
Welsh DK, Yoo SH, Liu AC et al (2004) Bioluminescence imaging of individual fibroblasts reveals persistent, independently phased circadian rhythms of clock gene expression. Curr Biol 14:2289–2295. doi:10.1016/j.cub.2004.11.057
Yamazaki S, Numano R, Abe M et al (2000) Resetting central and peripheral circadian oscillators in transgenic rats. Science 288:682–685. doi:10.1126/science.288.5466.682
Yoshikawa A, Shimada H, Numazawa K et al (2008) Establishment of human cell lines showing circadian rhythms of bioluminescence. Neurosci Lett 446:40–44. doi:10.1016/j.neulet.2008.08.091
Acknowledgements
This work was supported financially by Grants-in-Aid from the Ministry of Health, Labor, and Welfare, and from the Ministry of Education, Culture, Sports, Science, and Technology.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Shimada, H., Numazawa, K., Sasaki, T. et al. Introduction of tau Mutation into Cultured Rat1-R12 Cells by Gene Targeting, Using Recombinant Adeno-Associated Virus Vector. Cell Mol Neurobiol 29, 699–705 (2009). https://doi.org/10.1007/s10571-009-9389-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10571-009-9389-z