title: A new positive/negative selection scheme for precise BAC recombineering.
abstract: Recombineering technology allows the modification of large DNA constructs without using restriction enzymes, enabling the use of bacterial artificial chromosomes (BACs) in genetic engineering of animals and plants as well as in the studies of structures and functions of chromosomal elements in DNA replication and transcription. Here, we report a new selection scheme of BAC recombineering. A dual kanamycin and streptomycin selection marker was constructed using the kanamycin resistance gene and bacterial rpsL (+) gene. Recombination cassettes generated using this dual marker was used to make precise modifications in BAC constructs in a two-step procedure without leaving behind any unwanted sequences. The dual marker was first inserted into the site of modifications by positive selection of kanamycin resistance. In the second step, the counter-selection of streptomycin sensitivity resulted in the replacement of the dual marker with intended modified sequences. This method of BAC modification worked as efficiently as the previously reported galK method and provided a faster and more cost-effective alternative to the galK method.
authors.affiliation: Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine Hershey, Hershey, PA 17033, USA.
authors.forename: Shuwen
authors.initials: S
authors.lastname: Wang
authors.forename: Yuanjun
authors.initials: Y
authors.lastname: Zhao
authors.forename: Melanie
authors.initials: M
authors.lastname: Leiby
authors.forename: Jiyue
authors.initials: J
authors.lastname: Zhu
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005686:Galactokinase; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D007612:Kanamycin; D051379:Mice; D011995:Recombination, Genetic; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins / Q000235:genetics; D013307:Streptomycin; D019098:Telomerase / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins; C000717190:RpsI protein, E coli; C506766:RpsL protein, E coli; D007612:Kanamycin; D005686:Galactokinase; C509186:TERT protein, human; D019098:Telomerase; C509187:Tert protein, mouse; D013307:Streptomycin