title: A low-copy-number plasmid for retrieval of toxic genes from BACs and generation of conditional targeting constructs.
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.
authors.affiliation: School of Biological Sciences, Inje University, Gimhae, South Korea.
authors.forename: Giyoun
authors.initials: G
authors.lastname: Na
authors.forename: Andrew
authors.initials: A
authors.lastname: Wolfe
authors.forename: Chemyong
authors.initials: C
authors.lastname: Ko
authors.forename: Hyesook
authors.initials: H
authors.lastname: Youn
authors.forename: Young-Min
authors.initials: YM
authors.lastname: Lee
authors.forename: Sung June
authors.initials: SJ
authors.lastname: Byun
authors.forename: Iksoo
authors.initials: I
authors.lastname: Jeon
authors.forename: Yongbum
authors.initials: Y
authors.lastname: Koo
mesh_terms: D000818:Animals; D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics; D053595:Embryonic Stem Cells / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics; D012093:Replicon
chemical_list: D004247:DNA