title: Direct cloning of genomic DNA by recombinogenic targeting method using a yeast-bacterial shuttle vector, pClasper.
abstract: We have developed a method to clone genomic DNA selectively into a yeast-bacterial shuttle vector, pClasper, by recombinogenic targeting in yeast. A gene-specific pClasper targeting vector was constructed with small recombinogenic ends (500 bp) derived from flanking sequences of the genomic region to be cloned. Linearized, recombinogenic pClasper targeting vector and native genomic DNA were cotransformed into yeast. The gene of interest is selectively cloned by recombination between the recombinogenic ends in the targeting vector and homologous regions in the genomic DNA. Here we demonstrate direct cloning of a stably integrated Hoxc8-LacZ-Ura3 reporter gene construct from a mouse embryo fibroblast cell line and single-copy genes from total human genomic DNA. The frequency of capture of the recombinant insert was 0.05-3% of transformants. In contrast to previous reports, we were able to clone genomic DNA directly with a vector containing yeast autonomous replicating sequences. This approach provides a powerful method with which to clone and modify genes precisely for functional analysis.
authors.affiliation: Genaissance Pharmaceuticals Inc., New Haven, Connecticut 06511, USA. j.bhargava@genaissance.com
authors.forename: J
authors.initials: J
authors.lastname: Bhargava
authors.forename: C S
authors.initials: CS
authors.lastname: Shashikant
authors.forename: J L
authors.initials: JL
authors.lastname: Carr
authors.forename: H
authors.initials: H
authors.lastname: Juan
authors.forename: K L
authors.initials: KL
authors.lastname: Bentley
authors.forename: F H
authors.initials: FH
authors.lastname: Ruddle
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000235:genetics; D002460:Cell Line; D002876:Chromosomes, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004270:DNA, Circular / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D051379:Mice; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics; D047888:Receptors, Tumor Necrosis Factor, Type I; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014162:Transfection
chemical_list: D015703:Antigens, CD; D004270:DNA, Circular; D018343:Receptors, Adrenergic, beta-2; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I