title: An easy and versatile 2-step protocol for targeted modification and subcloning of DNA from bacterial artificial chromosomes using non-commercial plasmids.
abstract: BACKGROUND
  Promoter-specific expression of foreign DNA in transgenic organisms often relies on bacterial artificial chromosomes (BACs). This approach requires modification and subcloning of BAC-DNA by recombineering technologies in Escherichia coli. Most current protocols rely on commercial kits or isolation of BACs, their transfer between different host strains, and their restriction.
  
  
  RESULTS
  In this report we present a 2-step protocol for efficient modification and subcloning of DNA from bacterial artificial chromosomes using the non-commercial plasmids pKM208 and pTP223, distributed from addgene.com. A targeting cassette was successfully integrated into a BAC and 42 kb of this construct were subcloned. Both a plasmid-derived substrate with longer homology arms and a PCR-generated substrate with short homology arms (50 bp) were used for recombination. pKM208 and pTP223 contain all required genes for recombineering, but differ in their antibiotic resistance genes. This makes the system independent of the selection markers on the DNA molecules targeted for recombination.
  
  
  CONCLUSIONS
  The time and cost saving protocol presented here compares favorably to currently used systems. Using non-commercial plasmids, it allows targeted modification and cloning of large DNA (> 40 kb) fragments in vivo without restriction and ligation. Furthermore, both steps are performed in the same host eliminating the need to isolate BAC DNA and to use different bacterial strains.
authors.affiliation: Department of Neurogenetics, Institute of Biology and Environmental Sciences, Carl von Ossietzky University, Carl-von-Ossietzky-Strasse 9-11, 26129 Oldenburg, Germany. heiner.hartwich@uni-oldenburg.de
authors.forename: Heiner
authors.initials: H
authors.lastname: Hartwich
authors.forename: Hans Gerd
authors.initials: HG
authors.lastname: Nothwang
mesh_terms: D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005818:Genetic Engineering; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011995:Recombination, Genetic
chemical_list: D004247:DNA