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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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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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Abbreviations

Amp:

Ampicillin

AmpR:

Ampicillin resistance

BAC:

Bacterial artificial chromosome

Cam:

Chloramphenicol

CamR:

Chloramphenicol resistance

FNFL :

FRT-Neo-FRT-loxP

Kan:

Kanamycin

LB:

Luria-Bertani medium

LB+Amp:

LB medium containing ampicillin

LB+Cam:

LB medium containing chloramphenicol

LB+Kan:

LB medium containing kanamycin

LNL :

loxP-Neo-loxP

PCR:

Polymerase chain reaction

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Acknowledgments

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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Authors and Affiliations

  1. School of Biological Sciences, Inje University, Gimhae, 621-749, South Korea

    Giyoun Na & Yongbum Koo

  2. Division of Endocrinology, Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA

    Andrew Wolfe

  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

    CheMyong Ko

  4. Department of Bioscience and Biotechnology, Sejong University, Kwangjin gu, Seoul, 143-747, South Korea

    Hyesook Youn

  5. Department of Microbiology, College of Medicine, Chungbuk National University, Cheongju, Chungbuk, 361-763, South Korea

    Young-Min Lee

  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

    Young-Min Lee

  7. Animal Biotechnology Division, National Institute of Animal Science, Suwon, 441-706, South Korea

    Sung June Byun

  8. Research Planning Team, National Institute of Animal Science, Suwon, 441-706, South Korea

    Iksoo Jeon

Authors
  1. Giyoun Na
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  2. Andrew Wolfe
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  3. CheMyong Ko
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  8. Yongbum Koo
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Correspondence to Yongbum Koo.

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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

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  • DOI: https://doi.org/10.1007/s12033-012-9591-y

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