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A Low-Copy-Number Plasmid for Retrieval of Toxic Genes from BACs and Generation of Conditional Targeting Constructs |
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| Authors: | Giyoun Na Andrew Wolfe CheMyong Ko Hyesook Youn Young-Min Lee Sung June Byun Iksoo Jeon Yongbum Koo |
| Institution: | 1. School of Biological Sciences, Inje University, Gimhae, 621-749, South Korea 2. Division of Endocrinology, Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA 3. Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 3806 VMSBS, MC-002, 2001 South Lincoln Avenue, Urbana, IL, 61802, USA 4. Department of Bioscience and Biotechnology, Sejong University, Kwangjin gu, Seoul, 143-747, South Korea 5. Department of Microbiology, College of Medicine, Chungbuk National University, Cheongju, Chungbuk, 361-763, South Korea 6. Department of Animal, Dairy, and Veterinary Sciences, Utah Science Technology and Research (USTAR), College of Agriculture, Utah State University, Logan, UT, 84322-4815, USA 7. Animal Biotechnology Division, National Institute of Animal Science, Suwon, 441-706, South Korea 8. Research Planning Team, National Institute of Animal Science, Suwon, 441-706, South Korea |
| Abstract: | 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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