Abstract
The production of transgenic mice using small DNA constructs has been widely used for many years to investigate the regulation of gene activity. Small plasmid-based constructs (less than 20 kb) have been favored for a number of reasons, particularly the ease with which they can be manipulated and purified in large quantities. While this approach is powerful, there are some problems associated with the size of these transgenes. In particular, many of these small transgenes do not reproduce accurately the expression seen from the endogenous gene. For some genes the regulatory elements that control activity are located at a distance from the promoter and can be omitted from the transgene. These may be enhancers, repressors, boundary elements, or even locus control regions (LCRs), which are responsible for maintaining the correct spatial and temporal expression patterns of a number of genes, such as the globin clusters in mouse and humans (1). More important, small transgenes are susceptible to position effects from the chromatin environment in which they integrate, which often results in either ectopic expression (from trapping of nearby enhancers for other genes) or suppression of gene activity. Finally, small transgenes usually integrate in a multicopy tandem arrangement that does not accurately reflect the situation seen at the endogenous locus. There is growing evidence from studies in mouse and humans that the regulatory elements for many imprinted genes may be widely dispersed within “imprinted domains,” which may span hundreds of kilobases (2,3). Therefore, it is unlikely that analysis of small transgenes will provide much useful information concerning the expression or mechanism of imprinting for the majority of this unusual class of genes.
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References
Dillon, N. and Grosveld, F. (1993), Transcriptional regulation of multigene loci: multilevel control. TIG 9, 134–137.
Ainscough, J. F.-X., John, R., and Surani, M.A. (1998),Mechanism of imprinting on mouse distal chromosome 7. Gen. Res. 72 237–245.
Buiting, K., Saitoh, S., Gross, S., Dittrich, B., Schwartz, S., Nicholls, R.D. and Horsthemke, B. (1995) Inherited microdeletions in the Angelman and Prader-Willisyndromes define an imprinting centre on human chromosome 15. Nature Genet. 9, 395–400.
Yang, X. W., Model, P., and Heintz, N. (1997) Homologous recombination based modification in Esherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nature Biotechnol. 15, 1–7.
Ainscough, J. F.-X., Koide, T,, Tada, M., Barton, S., and Surani, M. A. (1997) Imprinting of Igf2 and H19 from a 130 kb YAC transgene. Development 124, 3621–3632.
Lee, J. T., Strauss, W. M., Dausman, J. A., and Jaenisch, R. (1996) A 450 kb transgene displays properties of the mammalian X-inactivation center. Cell 86, 83–94.
Schedl, A., Ross, A., Lee, M., Engelkamp, D., Rashbass, P., van Heyningen, V., and Hastie, N. D. (1996) Influence of PAX6 gene dosage on development: overexpression causes severe eye abnormalities. Cell 86, 71–82.
Wutz, A., Smrzka, O. W., Wagner, E. F., and Barlow, D. P. (1997) Imprinted expression of the Igf2r gene depends on an intronic Cpp island. Nature 389, 745–749.
Rothstein, R. (1991) Targeting, disruption, replacement and allele rescue: integrative DNA transformation in yeast, in Guide to Yeast Genetics and Molecular Biology (Guthrie, C., and Fink, G., eds.), Academic, San Diego, CA, 281–301.
Ainscough, J. F.-X., John, R. M., Barton, S. C., and Surami, M. A. (2000) A skeletal muscle-specific mouse IGF2 repressor lies 40Kb downstream of the gene. Development 127, 3923–3930.
Schedl, A., Grimes, B., and Montoliu, L. (1996) YAC transfer by microinjection. Meth. Mol. Biol. 293–306.
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© 2002 Humana Press Inc., Totowa, NJ
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Ainscough, J.FX., John, R.M., Barton, S.C. (2002). Production of YAC Transgenic Mice by Pronuclear Injection. In: Ward, A. (eds) Genomic Imprinting. Methods in Molecular Biology™, vol 181. Humana Press. https://doi.org/10.1385/1-59259-211-2:55
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DOI: https://doi.org/10.1385/1-59259-211-2:55
Publisher Name: Humana Press
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