Abstract
Since their first description in 1991 (1), CAG-disease causing genes are increasing in number. Up to date, there are at least nine genetic diseases caused by CAG expansions. The creation of transgene and knock-in mice with CAG expansions is an useful tool for understanding the pathological mechanisms of the corresponding diseases, which could lead to therapeutic target(s) for the diseases. However, owing to the short life expectancy of the mice and to the low expression levels of transgenes, it is necessary to introduce CAG expansions larger than that in humans in order to elicit a pathological phenotype within the life of mice models. Naturally occurring “huge” CAG expansions (>100-150 CAGs), which could induce disease phenotype in the mice, are very seldom. In vitro synthesis of isolated CAG repeats have already been described (2,3). Most of these methods, however, require further cloning steps and often contain some flanking extraneous sequences. Here, the author describes a fast and simple way for expanding/introducing CAG repeats (or other repeats!) without altering the flanking 5′ and 3′ sequences of the gene of interest. This method was successfully employed for expanding the CAG repeat of the MJD/SCA3 gene (4). Fig. 1 outlined the strategy of this method. Two independent polymerase chain reactions (PCRs) amplify the target gene from 5′ to the CAG repeat region (PCR I) and from the CAG repeat to the 3′ region (PCR II) of the gene. The amplicons of PCR I and PCR II will be then mixed, elongated and then a third PCR will be carried out with the two most “outsider” primers. We used this strategy for elongating the CAG of the MJD/SCA3 gene from 22 up to 138 CAG repeats (see Figs. 2and3).
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References
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© 2002 Humana Press Inc.
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Laccone, F. (2002). A Fast Polymerase Chain Reaction-Mediated Strategy for Introducing Repeat Expansions into CAG-Repeat Containing Genes. In: Chen, BY., Janes, H.W. (eds) PCR Cloning Protocols. Methods in Molecular Biology™, vol 192. Humana Press. https://doi.org/10.1385/1-59259-177-9:217
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DOI: https://doi.org/10.1385/1-59259-177-9:217
Publisher Name: Humana Press
Print ISBN: 978-0-89603-969-8
Online ISBN: 978-1-59259-177-0
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