Abstract
The ability to efficiently and accurately modify genomic DNA through targeted and tissue-specific mutations is an important goal in animal transgenesis. Here we describe how to exploit two systems of homologous recombination, from yeast and bacteria, to engineer yeast artificial chromosomes (YACs) to generate targeted deletions and inversions in vivo, in transgenic animals, and in the presence of DNA-modifying enzymes known as recombinases. Through homologous recombination in yeast, specific recombinogenic sequences are inserted upstream and downstream of a region in the YAC. The sites of integration of these short sequence elements are chosen carefully, such that the YAC is left functionally intact, and this modified transgene represents the wild-type allele. This YAC is subsequently used to generate transgenic animals, which when bred to animals expressing recombinase proteins result in genetic modifications. By expressing recombinase proteins from different tissue-specific promoters, one can mediate site-specific recombination to generate either ubiquitous or tissue-specific deletions or inversion. These modifications can then be carried through the germline or can be studied somatically. A great advantage of this system is the ability to evaluate subtle genetic effects independent of position-effect variegation, and transgene copy number, eliminating the need to examine several independently generated lines of transgenic animals for each genetic variant.
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References
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. R., eds.), Academic, San Diego, CA, pp. 281–301.
Sikorski, R. S. and Boeke, J. D. (1991) In vitro mutagenesis and plasmid shuffling: from cloned gene to mutated yeast. In: Guide to Yeast Genetics and Molecular Biology (Guthrie, C. and Fink, G. R., eds.), Academic, San Diego, CA, pp. 302–318.
Srivastava, A. K. and Schlessinger, D. (1991) Vectors for inserting selectable markers in vector arms and human DNA inserts of yeast artificial chromosomes (YACs). Gene 103, 53–59.
Sauer, B. and Henderson, N. (1988) Site specific DNA recombination in mammalian cells by Cre recombinase of bacteriophage P1. PNAS 85, 5166–5170.
Gronostajski, R. M. and Sadowski, P. D. (1985) Determination of DNA sequences essential for FLP-mediated recombination by a novel method. J. Biol. Chem. 260, 12,320–12,327.
Peterson, K. R., Clegg, C. H., Huxley, C., et al. (1993) Transgenic mice containing a 248-kb yeast artificial chromosome carrying the human β-globin locus display proper developmental control of human globin genes. PNAS 90, 7593–7597.
Loots, G. G., Locksley, R. M., Blankespoor, C. M., et al. (2000) Identification of a coordinate regulator of interleukins 4, 13, and 5 by cross-species sequence comparisons. Science 288, 136–140.
Pachnis, V., Pevny, L., Rothstein, R., and Constantini, F. (1990) Transfer of a yeast artificial chromosome carrying human DNA from Saccharomyces cerevisiae into mammalian cells. PNAS 87, 5109–5113.
Cook, J. R., Emanuel, S. L., and Pestka, S. (1993) Yeast artificial chromosome fragmentation vectors that utilize URA3 selection. Genet. Anal. Tech. Appl. 10, 109–112.
Srivastava, A. K. and Schlessinger, D. (1991) Vectors for inserting selectable markers in vector arms and human DNA inserts of yeast artificial chromosomes (YACs). Gene 103, 53–59.
Markie, D., Ragoussis, J., Senger, G., et al. (1993) New vector for transfer of yeast aritificial chromosomes to mammalian cells. Somat. Cell Mol. Genet. 19, 161–169.
Davies, N. P., Rosewell, I. R., and Bruggemann, M. (1992) Targeted alterations in yeast artificial chromosomes for inter-species gene transfer. NAR 20, 2693–2698.
Green, E. D. and Olson, M. V. (1990) Chromosomal region of the cystic fibrosis gene in yeast artificial chromosomes: a model for human genome mapping. Science 250, 94–98.
Spencer, F., Hugerat, Y., Simchen, G., Hurko, O., Connelly, C., and Hieter, P. (1994) Yeast kar1 mutants provide an effective method for YAC transfer to new hosts. Genomics 22, 118–126.
Connelly, C., McCormick, M., Shero, J., and Hieter, P. (1991) Polyamines eliminate an extreme size bias against transformation of large yeast artificial chromosome DNA. Genomics 10, 10–16.
Sikorski, R. S. and Hieter, P. (1989) A system of shuttle vectors and yeast host strains designated for efficient manipulation of DNA in S. cerevisiae. Genetics 122, 19–27.
Sakai, K. and Miyazaki, J. (1997) A transgenic mouse line that retains Cre recombinase activity in mature oocytes irrespective of the Cre transgene transmission. Biochem. Biophys. Res. Commun. 237, 318–324.
Heidt, A. B. and Black B. L. (2005) Transgenic mice that express Cre recombinase under control of a skeletal muscle-specific promoter from mef2c. Genesis 42, 28–32.
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© 2006 Humana Press Inc., Totowa NJ
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Loots, G.G. (2006). Modifying Yeast Artificial Chromosomes to Generate Cre/LoxP and FLP/FRT Site-Specific Deletions and Inversions. In: MacKenzie, A. (eds) YAC Protocols. Methods in Molecular Biology™, vol 349. Humana Press. https://doi.org/10.1385/1-59745-158-4:75
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DOI: https://doi.org/10.1385/1-59745-158-4:75
Publisher Name: Humana Press
Print ISBN: 978-1-58829-612-2
Online ISBN: 978-1-59745-158-1
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