Abstract
Bacterial artificial chromosome (BAC) has the capacity to clone DNA fragments in excess of 300 kb. It also has the considerable advantages of stable propagation and ease of purification. These features make BAC suitable in genetic research, such as library construction, transgenic mice production, and gene targeting constructs. Homologous recombination in Escherichia coli, a process named recombineering, has made the modification of BACs easy and reliable. We report here a modified recombineering method that can efficiently mediate the fusion of large DNA fragments from two or more different BACs. With the introduction of kanamycin-resistant gene and proposed rare-cutting restriction endonuclease (RCRE) sites into two BACs, a 82.6-kb DNA frament containing the inverted human α-globin genes (ϑ, α1, α2, and ζ) from BAC191K2 and the locus control region (LCR) of human β-globin gene locus (from the BAC186D7) was reconstructed. This approach for combining different BAC DNA fragments should facilitate many kinds of genomic experiments.
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References
Heintz, N. (2001) BAC to the future: the use of bac transgenic mice for neuroscience research. Nat. Rev. Neurosci. 2, 861–870.
Gong, S., Zheng, C., Doughty, M. L., et al. (2003) A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature 425, 917–925.
Boren, J., Lee, I., Callow, M. J., Rubin E. M., and Innerarity, T. L. (1996) A simple and efficient method for making site-directed mutants, deletions, and fusions of large DNA such as P1 and BAC clones. Genome Res. 6, 1123–1130.
Yang, X. W., Wynder, C., Doughty M. L., and Heintz, N. (1999) BAC-mediated gene-dosage analysis reveals a role for Ziprol (Ru49/Zfp38) in progenitor cell proliferation in cerebellum and skin. Nat. Genet. 22, 327–335.
Muyrers, J. P., Zhang, Y., Testa, G., and Stewart, A. F. (1999) Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res. 27, 1555–1557.
Lee, E. C., Yu, D., and Martinez, de Velasco J. (2001) A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73, 56–65.
Muyrers, J. P., Zhang, Y., and Benes, V. (2000) Point mutation of bacterial artificial chromosomes by ET recombination. EMBO Rep. 1, 239–243.
Testa, G., Zhang, Y., Vintersten, K., et al. (2003) Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles. Nat. Biotechnol. 21, 443–447.
Yang, Y. and Seed, B. (2003) Site-specific gene targeting in mouse embryonic stem cells with intact bacterial artificial chromosomes. Nat. Biotechnol. 21, 447–451.
Zhang, X. M. and Huang, J. D. (2003) Combination of overlapping bacterial artificial chromosomes by a two-step recombinogenic engineering method. Nucleic Acids Res. 31, e81.
Huang, Y., Liu, D. P., Wu, L., et al. (2000) Proper developmental control of human globin genes reproduced by transgenic mice containing a 160-kb BAC carrying the human beta-globin locus. Blood Cells Mol. Dis. 26, 598–610.
Feng, D. X., Liu, D. P., Huang, Y., et al. (2001) The expression of human alpha-like globin genes in transgenic mice mediated by bacterial artificial chromosome. Proc. Natl. Acad. Sci. USA 98, 15,073–15,077.
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Shen, W., Huang, Y., Tang, Y. et al. A general method to modify BACs to generate large recombinant DNA fragments. Mol Biotechnol 31, 181–186 (2005). https://doi.org/10.1385/MB:31:3:181
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DOI: https://doi.org/10.1385/MB:31:3:181