title: Bacterial delivery of large intact genomic-DNA-containing BACs into mammalian cells.
abstract: Efficient delivery of large intact vectors into mammalian cells remains problematical. Here we evaluate delivery by bacterial invasion of two large BACs of more than 150 kb in size into various cells. First, we determined the effect of several drugs on bacterial delivery of a small plasmid into different cell lines. Most drugs tested resulted in a marginal increase of the overall efficiency of delivery in only some cell lines, except the lysosomotropic drug chloroquine, which was found to increase the efficiency of delivery by 6-fold in B16F10 cells. Bacterial invasion was found to be significantly advantageous compared with lipofection in delivering large intact BACs into mouse cells, resulting in 100% of clones containing intact DNA. Furthermore, evaluation of expression of the human hypoxanthine phosphoribosyltransferase (HPRT) gene from its genomic locus, which was present in one of the BACs, showed that single copy integrations of the HPRT-containing BAC had occurred in mouse B16F10 cells and that expression of HPRT from each human copy was 0.33 times as much as from each endogenous mouse copy. These data provide new evidence that bacterial delivery is a convenient and efficient method to transfer large intact therapeutic genes into mammalian cells.
authors.affiliation: Division of Natural Sciences, Imperial College London, London, UK.
authors.forename: Wing
authors.initials: W
authors.lastname: Cheung
authors.forename: George
authors.initials: G
authors.lastname: Kotzamanis
authors.forename: Hassan
authors.initials: H
authors.lastname: Abdulrazzak
authors.forename: Sylvie
authors.initials: S
authors.lastname: Goussard
authors.forename: Tadashi
authors.initials: T
authors.lastname: Kaname
authors.forename: Athanassios
authors.initials: A
authors.lastname: Kotsinas
authors.forename: Vassilis G
authors.initials: VG
authors.lastname: Gorgoulis
authors.forename: Catherine
authors.initials: C
authors.lastname: Grillot-Courvalin
authors.forename: Clare
authors.initials: C
authors.lastname: Huxley
mesh_terms: D000818:Animals; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010957:Plasmids
chemical_list: D007041:Hypoxanthine Phosphoribosyltransferase
references.citation: Proc Natl Acad Sci U S A. 1978 Jul;75(7):3327-31
references.pmid: 28524
references.citation: Gene Ther. 2005 Nov;12(21):1559-72
references.pmid: 15973438
references.citation: Curr Opin Biotechnol. 1999 Oct;10(5):477-81
references.pmid: 10508638
references.citation: Hum Mol Genet. 1998 Mar;7(3):449-58
references.pmid: 9467003
references.citation: Nat Biotechnol. 1998 Sep;16(9):862-6
references.pmid: 9743121
references.citation: Genomics. 2007 Nov;90(5):610-9
references.pmid: 17822869
references.citation: Mamm Genome. 2004 May;15(5):370-82
references.pmid: 15170226
references.citation: J Cell Biol. 1979 Sep;82(3):644-53
references.pmid: 511930
references.citation: J Cell Mol Med. 2009 Sep;13(9A):2938-48
references.pmid: 18657227
references.citation: Cell Death Differ. 2005 Nov;12(11):1439-41
references.pmid: 15962008
references.citation: J Gene Med. 2004 Feb;6(2):195-209
references.pmid: 14978773
references.citation: Hum Mol Genet. 2002 May 1;11(9):1075-94
references.pmid: 11978767
references.citation: Nucleic Acids Res. 1996 Dec 1;24(23):4693-9
references.pmid: 8972855
references.citation: BMC Biotechnol. 2003 Feb 03;3(1):2
references.pmid: 12609052
references.citation: Blood. 2001 Oct 15;98(8):2396-402
references.pmid: 11588036
references.citation: Cell Microbiol. 2001 Sep;3(9):599-609
references.pmid: 11553012
references.citation: Gene Ther. 1996 Sep;3(9):797-801
references.pmid: 8875228
references.citation: J Cell Biol. 1981 Sep;90(3):656-64
references.pmid: 7287819
references.citation: Gene. 2001 Mar 21;266(1-2):147-53
references.pmid: 11290429
references.citation: Int J Pharm. 2001 May 7;218(1-2):81-92
references.pmid: 11337152
references.citation: J Cell Biol. 1983 Dec;97(6):1841-51
references.pmid: 6196369
references.citation: Hum Mol Genet. 2000 Mar 1;9(4):617-29
references.pmid: 10699185
references.citation: J Cell Biol. 1981 Sep;90(3):665-9
references.pmid: 6169733
references.citation: J Biol Chem. 1994 Apr 29;269(17):12918-24
references.pmid: 8175709
references.citation: J Gene Med. 2005 Aug;7(8):1097-112
references.pmid: 16001391