title: Towards genetic genome projects: genomic library screening and gene-targeting vector construction in a single step.
abstract: We have developed technologies that simplify genomic library construction and screening, substantially reducing both the time and the cost associated with traditional library screening methods and facilitating the generation of gene-targeting constructs. By taking advantage of homologous recombination in Escherichia coli, we were able to use as little as 80 bp of total sequence homology to screen for a specific gene from a genomic library in plasmid or phage form. This method, called recombination cloning (REC), takes only a few days instead of the several weeks required for traditional plaque-lift methods. In addition, because every clone in the mouse genomic library we have constructed has a negative selection marker adjacent to the genomic insert, REC screening can generate gene-targeting vectors in one step, from library screening to finished construct. Conditional targeting constructs can be generated easily with minimal additional manipulation.
authors.affiliation: Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: Pumin
authors.initials: P
authors.lastname: Zhang
authors.forename: Mamei Z
authors.initials: MZ
authors.lastname: Li
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Elledge
mesh_terms: D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004268:DNA-Binding Proteins; D015252:Deoxyribonucleases, Type II Site-Specific; D004351:Drug Resistance / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics; D005240:Feasibility Studies; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D015723:Gene Library; D018390:Gene Targeting / Q000379:methods*; D005796:Genes; D005822:Genetic Vectors / Q000235:genetics*; D016678:Genome; D016680:Genome, Bacterial; D023281:Genomics / Q000191:economics / Q000379:methods*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008297:Male; D051379:Mice; D009363:Neoplasm Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins; D064249:Securin; D012641:Selection, Genetic; D012689:Sequence Homology, Nucleic Acid; D000198:Spectinomycin / Q000494:pharmacology; D014764:Viral Proteins / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D029701:Saccharomyces cerevisiae Proteins; D064249:Securin; D014764:Viral Proteins; C065410:gam protein, Coliphage; D007544:Isopropyl Thiogalactoside; D000198:Spectinomycin; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific