title: DNA modification and functional delivery into human cells using Escherichia coli DH10B.
abstract: The availability of almost the complete human genome as cloned BAC libraries represents a valuable resource for functional genomic analysis, which, however, has been somewhat limited by the ability to modify and transfer this DNA into mammalian cells intact. Here we report a novel comprehensive Escherichia coli-based vector system for the modification, propagation and delivery of large human genomic BAC clones into mammalian cells. The GET recombination inducible homologous recombination system was used in the BAC host strain E.coli DH10B to precisely insert an EGFPneo cassette into the vector portion of a approximately 200 kb human BAC clone, providing a relatively simple method to directly convert available BAC clones into suitable vectors for mammalian cells. GET recombination was also used for the targeted deletion of the asd gene from the E.coli chromosome, resulting in defective cell wall synthesis and diaminopimelic acid auxotrophy. Transfer of the Yersinia pseudotuberculosis invasin gene into E.coli DH10B asd(-) rendered it competent to invade HeLa cells and deliver DNA, as judged by transient expression of green fluorescent protein and stable neomycin-resistant colonies. The efficiency of DNA transfer and survival of HeLa cells has been optimized for incubation time and multiplicity of infection of invasive E.coli with HeLa cells. This combination of E.coli-based homologous recombination and invasion technologies using BAC host strain E.coli DH10B will greatly improve the utility of the available BAC libraries from the human and other genomes for gene expression and functional genomic studies.
authors.affiliation: Department of Human Genetics, Box 1498, Mount Sinai School of Medicine, 1425 Madison Avenue, East Building 14-52A, New York, NY 10029, USA.
authors.forename: Kumaran
authors.initials: K
authors.lastname: Narayanan
authors.forename: Peter E
authors.initials: PE
authors.lastname: Warburton
mesh_terms: D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D004247:DNA / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D049452:Green Fluorescent Proteins; D006367:HeLa Cells / Q000382:microbiology*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008858:Microscopy, Phase-Contrast; D010957:Plasmids / Q000235:genetics; D014162:Transfection / Q000379:methods
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004247:DNA