Abstract
The genomic DNA libraries based on Bacteria Artificial Chromosomes (BAC) are the foundation of whole genomic mapping, sequencing, and annotation for many species like mice and humans. With their large insert size, BACs harbor the gene-of-interest and nearby transcriptional regulatory elements necessary to direct the expression of the gene-of-interest in a temporal and cell-type specific manner. When replacing a gene-of-interest with a transgene in vivo, the transgene can be expressed with the same patterns and machinery as that of the endogenous gene. This chapter describes in detail a method of using lambda-red recombineering to make BAC transgene constructs with the integration of a transgene into a designated location within a BAC. As the final BAC construct will be used for transfection in cell lines or making transgenic animals, specific considerations with BAC transgenes such as genotyping, BAC coverage and integrity as well as quality of BAC DNA will be addressed. Not only does this approach provide a practical and effective way to modify large DNA constructs, the same recombineering principles can apply to smaller high copy plasmids as well as to chromosome engineering.
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Acknowledgements
The authors thank Dr. B.L. Wanner of Purdue University for the pKD46 plasmid, which was distributed by the E. Coli Genetic Stock Center at Yale. The authors also thank Dr. N. Copeland for providing DY380 and EL250/350. The protocol described here has been used in the UNC Neuroscience Center’s BAC Engineering Core and is supported by an NINDS Center Grant.
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Holmes, S., Lyman, S., Hsu, JK., Cheng, J. (2015). Making BAC Transgene Constructs with Lambda-Red Recombineering System for Transgenic Animals or Cell Lines. 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_4
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DOI: https://doi.org/10.1007/978-1-4939-1652-8_4
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