Abstract
Recombineering technology allows the modification of large DNA constructs without using restriction enzymes, enabling the use of bacterial artificial chromosomes (BACs) in genetic engineering of animals and plants as well as in the studies of structures and functions of chromosomal elements in DNA replication and transcription. Here, we report a new selection scheme of BAC recombineering. A dual kanamycin and streptomycin selection marker was constructed using the kanamycin resistance gene and bacterial rpsL + gene. Recombination cassettes generated using this dual marker was used to make precise modifications in BAC constructs in a two-step procedure without leaving behind any unwanted sequences. The dual marker was first inserted into the site of modifications by positive selection of kanamycin resistance. In the second step, the counter-selection of streptomycin sensitivity resulted in the replacement of the dual marker with intended modified sequences. This method of BAC modification worked as efficiently as the previously reported galK method and provided a faster and more cost-effective alternative to the galK method.
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Acknowledgments
DY380 recombineering system was kindly provided by the Copeland laboratory at National Cancer Institute, Frederick, MD. We thank Dr. Renjith Mathew for critical review of the manuscript. The study was supported in part by NIH grant GM071725. JZ is a Research Scholar of American Cancer Society. The authors declare no competing interests.
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Wang, S., Zhao, Y., Leiby, M. et al. A New Positive/Negative Selection Scheme for Precise BAC Recombineering. Mol Biotechnol 42, 110–116 (2009). https://doi.org/10.1007/s12033-009-9142-3
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DOI: https://doi.org/10.1007/s12033-009-9142-3