A low-copy-number plasmid for retrieval of toxic genes from BACs and generation of conditional targeting constructs

Giyoun Na, Andrew Wolfe, Chemyong Ko, Hyesook Youn, Young Min Lee, Sung June Byun, Iksoo Jeon, Yongbum Koo

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)504-514
Number of pages11
JournalMolecular Biotechnology
Volume54
Issue number2
DOIs
StatePublished - Jun 2013

Keywords

  • Bacterial artificial chromosome
  • Low-copy-number plasmid
  • Recombineering
  • Targeting vector
  • Toxic gene

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology

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