Summary
This chapter describes how transgenic mice can be made with human genomic DNA fragments cloned from DM1 patients’ DNA and how the CTG repeat instability is assessed over generations and in different tissues. Construction of cosmid libraries is fully reported from the extraction of high-molecular-weight DNA from patients’ lymphoid cell lines, to the screening and mapping of the positive clones. After establishment of transgenic lines, we explained the methods used to analyze (a) the CTG repeats that are inherited from the transgenic parents, with regard to age, sex, and parental CTG repeat sizes, and (b) the CTG repeat-length variations that can be observed in somatic tissues and in sperm.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Harper, P. S. (2001) Myotonic Dystrophy, 3rd ed. Saunders, London.
Aslanidis, C., Jansen, G., Amemiya, C., et al. (1992) Cloning of the essential myotonic dystrophy region and mapping of the putative defect. Nature 355, 548–551.
Brook, J. D., McCurrach, M. E., Harley, H. G., et al. (1992) Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase family member. Cell 68, 799–808.
Fu, Y. H., Kuhl, D. P. A., Pizzuti, A., et al. (1992) An unstable triplet repeat in a gene related to myotonic muscular dystrophy. Science 255, 1256–1258.
Mahadevan, M., Tsilfidis, C., Sabourin, L., et al. (1992) Myotonic dystrophy mutation: an unstable CTG repeat in the 3′ untranslated region of the gene. Science 255, 1253–1255.
Gourdon, G., Dessen, P., Lia, A. S., et al. (1997) Intriguing association between disease associated unstable trinucleotide repeat and CpG island. Ann. Genet. 40, 73–77.
Seznec, H., Lia-Baldini, A., Duros, C., et al. (2000) Transgenic mice carrying large human genomic sequences with expanded CTG repeat mimic closely the DM CTG repeat intergenerational and somatic instability. Hum. Mol. Genet. 9, 1185–1194.
Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY I.126–I.142.
Hogan, B., Beddington, R., Costantini, F., et al. (1994) Manipulating the Mouse Embryo: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Jeffreys, A., Neumann, R., and Wilson, V. (1990). Repeat unit sequence variation in minisatellites: a novel source of polymorphism for studying allelic variation and mutation by single molecule analysis. Cell 60, 473–485.
Monckton, D. G., Wong, L. J. C., Ashizawa, T., et al. (1995) Somatic mosaicism, germline expansions, germline reversions and intergenerational reductions in myotonic dystrophy males: small pool PCR analyses. Hum. Mol. Genet. 4, 1–8.
Jeffreys, A. J., Tamaki, K., MacLeod, A., et al. (1994) Complex gene conversion events in germline mutation at human minisatellites. Nature Genet. 6, 136–145.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc.
About this protocol
Cite this protocol
Savouret, C., Junien, C., Gourdon, G. (2004). Analysis of CTG Repeats Using DM1 Model Mice. In: Kohwi, Y. (eds) Trinucleotide Repeat Protocols. Methods in Molecular Biology™, vol 277. Humana Press. https://doi.org/10.1385/1-59259-804-8:185
Download citation
DOI: https://doi.org/10.1385/1-59259-804-8:185
Publisher Name: Humana Press
Print ISBN: 978-1-58829-243-8
Online ISBN: 978-1-59259-804-5
eBook Packages: Springer Protocols