Towards genetic genome projects: genomic library screening and gene-targeting vector construction in a single step.
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.
Zhang
Pumin
P
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030, USA.
Li
Mamei Z
MZ
Elledge
Stephen J
SJ
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
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