Abstract
In the era of functional genomics, bacterial artificial chromosome (BAC) is an ideal choice of vector for cloning and manipulating large DNA fragments (1). In addition to providing a system that is able to maintain large DNA fragments (average insert size approx 150–200 kb), BAC DNA can be purified by using standard plasmid purification method. Functional analysis of the BAC insert, which may represent coding or regulatory sequences or an uncharacterized DNA fragment, may require manipulation of the BAC DNA. For example, a drug resistance marker may have to be inserted to enable selection of the BAC DNA when transfected into eukaryotic cells, a gene present in the BAC insert may have to be mutated for functional studies or a transgene may have to be introduced for ectopic expression. Depending on the goal, the method of manipulation is selected. BACs are maintained in Escherichia coli (E. coli) cells that are recA − to ensure the stability of the insert. However, this hinders manipulation of BACs by homologous recombination in the bacterial cells. Consequently, the gene(s) required for recombination is transiently expressed to facilitate homologous recombination. Several different systems have been developed for BAC engineering, each utilizing a different protein(s) (such as recA, recE and recT, or exo and bet), to enable the BACs to undergo recombination (2–5).
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References
Shizuya, H., Birren B., Kim, U. J., et al. (1992) qCloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vectorai. Proc. Natl. Acad. Sci. USA 89, 8794–8797.
Yang, X. W., Model, P., and Heintz, N. (1997) Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nature Biotech. 15, 859–865.
Zhang, Y., Buchholz, F., Muyrers, J. P. P., and Stewart, A. F. (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nature Genet. 20, 123–128.
Muyrers, J. P. P., Zhang, Y., Testa, G., and Stewart, A. F. (1999) Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucl. Acids Res. 27, 1555–1557.
Yu, D., Ellis, H. M., Lee, E.-C., Jenkins, N. A., Copeland, N. G., and Court, D. L. (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. USA 97, 5978–5983.
Lee, E.-C., Yu, D., Martinez de Velasco, J., et al. (2001) A Highly Efficient Escherichia coli-Based Chromosome Engineering System Adapted for Recombinogenic Targeting and Subcloning of BAC DNA. Genomics 73, 56–65.
Sambrook, J., Fritch, E. F., and Maniatis, T. (1989) Molecular Cloning: A laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Sinnett, D., Richer, C., and Baccichet, A. (1998) Isolation of stable bacterial artificial chromosome DNA using a modified alkaline lysis method. Biotech. 24, 752–754.
Blomfield, I. C., Vaughn, V., Rest, R. F., and Eisenstein, B. I. (1991) Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon. Mol. Microbiol. 5, 1447–1457.
Swaminathan, S., Ellis, H. M., Waters, L. S., et al. (2001) Rapid engineering of bacterial artificial chromosomes using oligonucleotides. Genesis 29, 14–21.
Cha, R. S., Zarbl, H., Keohavong, P., and Thilly, W. G. (1992) Mismatch amplification mutation assay (MAMA): Application to the c-H-ras gene. PCR Meth. Appl. 2, 14–20.
Court, D. L., Swaminathan, S., Yu, D., et al. (2003) Mini-lambda: a tractable system for chromosome and BAC enginering. Gene 315, 63–69.
Yang, Y., Sharan, S. K. (2003) A simple two-step, ‘hit and fix’ method to generate subtle mutations in BACs using short denatured PCR fragments. Nucleic Acids Res. 31, e80.
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© 2004 Humana Press Inc., Totowa, NJ
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Swaminathan, S., Sharan, S.K. (2004). Bacterial Artificial Chromosome Engineering. In: Zhao, S., Stodolsky, M. (eds) Bacterial Artificial Chromosomes. Methods in Molecular Biology, vol 256. Humana Press. https://doi.org/10.1385/1-59259-753-X:089
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DOI: https://doi.org/10.1385/1-59259-753-X:089
Publisher Name: Humana Press
Print ISBN: 978-0-89603-989-6
Online ISBN: 978-1-59259-753-6
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