Bacterial Artificial Chromosome (BAC) clones are widely used for retrieving genomic DNA sequences for gene targeting. In this study, low-copy-number plasmids pBAC-FB, pBAC-FC, and pBAC-DE, which carry the F plasmid replicon, were generated from pBACe3.6. pBAC-FB was successfully used to retrieve a sequence of a BAC that was resistant to retrieval by a high-copy-number plasmid via λ Red-mediated recombineering (gap-repair cloning). This plasmid was also used to retrieve two other genes from BAC, indicating its general usability retrieving genes from BAC. The retrieved genes were manipulated in generating targeting vectors for gene knockouts by recombineering. The functionality of the targeting vector was further validated in a targeting experiment with C57BL/6 embryonic stem cells. The low-copy-number plasmid pBAC-FB is a plasmid of choice to retrieve toxic DNA sequences from BACs and to manipulate them to generate gene-targeting constructs by recombineering.
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Bacterial artificial chromosome
- FNFL :
LB medium containing ampicillin
LB medium containing chloramphenicol
LB medium containing kanamycin
- LNL :
Polymerase chain reaction
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This work was supported by a grant from Inje Research and Scholarship Foundation in 2012, by a grant from Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2011-0011173), and by a grant UO1 HD066432 from National Institutes of Health.
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Na, G., Wolfe, A., Ko, C. et al. A Low-Copy-Number Plasmid for Retrieval of Toxic Genes from BACs and Generation of Conditional Targeting Constructs. Mol Biotechnol 54, 504–514 (2013). https://doi.org/10.1007/s12033-012-9591-y