Abstract
BAC clones containing the entire genomic region of a gene including the long-range regulatory elements are very useful for gene functional analysis. However, large genes often span more than the insert of a BAC clone, and single BACs covering the entire region of interest are not available. Here, we describe a general system for linking two or more overlapping BACs into a single clone. Two rounds of homologous recombination are used. In the first, the BAC inserts are subcloned into the pBACLink vectors. In the second, the two BACs are combined together. Multiple BACs in a contig can be combined by alternating use of the pBACLInk vectors, resulting in several BAC clones containing as much of the genomic region of a gene as required. Such BACs can then be used in gene expression studies and/or gene therapy applications.
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References
Osoegawa K, Mammoser AG, Wu C et al (2001) A bacterial artificial chromosome library for sequencing the complete human genome. Genome Res 11(3):483–496
Copeland NG, Jenkins NA, Court DL (2001) Recombineering: a powerful new tool for mouse functional genomics. Nat Rev Genet 2:769–779
Muyrers JPP, Zhang Y, Stewart AF (2001) Techniques: recombinogenic engineering – new options for cloning and manipulating DNA. Trends Biochem Sci 26:327–331
Yu D, Ellis HM, Lee EC et al (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U S A 97:5978–5983
Lee EC, Yu D, de Velasco JM 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
Kotzamanis G, Huxley C (2004) Recombining overlapping BACs into a single larger BAC. BMC Biotechnol 4:1
Kotzamanis G, Abdulrazzak H, Gifford-Garner J et al (2009) CFTR expression from a BAC carrying the complete human gene and associated regulatory elements. J Cell Mol Med 13(9A):2938–2948
Kim UJ, Birren BW, Slepak T et al (1996) Construction and characterization of a human bacterial artificial chromosome library. Genomics 34:213–218
Acknowledgement
Financial support was from the European Commission FP7 project INsPiRE.
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Kotzamanis, G., Kotsinas, A. (2015). Recombining Overlapping BACs into Single Large BACs. In: Narayanan, K. (eds) Bacterial Artificial Chromosomes. Methods in Molecular Biology, vol 1227. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1652-8_6
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DOI: https://doi.org/10.1007/978-1-4939-1652-8_6
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1651-1
Online ISBN: 978-1-4939-1652-8
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