Delivery of bacterial artificial chromosomes into mammalian cells with psoralen-inactivated adenovirus carrier.
Molecular biology has many applications where the introduction of large (>100 kb) DNA molecules is required. The current methods of large DNA transfection are very inefficient. We reasoned that two limits to improving transfection methods with these large DNA molecules were the difficulty of preparing workable quantities of clean DNA and the lack of rapid assays to determine transfection success. We have used bacterial artificial chromosomes (BACs) based on the Escherichia coli F factor plasmid system, which are simple to manipulate and purify in microgram quantities. Because BAC plasmids are kept at one to two copies per cell, the problems of rearrangement observed with YACs are eliminated. We have generated two series of BAC vectors bearing marker genes for luciferase and green fluorescent protein (GFP). Using these reagents, we have developed methods of delivering BACs of up to 170 kb into mammalian cells with transfection efficiency comparable to 5-10 kb DNA. Psoralen-inactivated adenovirus is used as the carrier, thus eliminating the problems associated with viral gene expression. The delivered DNA is linked to the carrier virus with a condensing polycation. Further improvements in gene delivery were obtained by replacing polylysine with low molecular weight polyethylenimine (PEI) as the DNA condensing agent.
Baker
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Institute for Molecular Pathology (IMP), Dr BohrGasse 7, 1030 Vienna, Austria.
Cotten
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D000256:Adenoviridae; D000818:Animals; D002460:Cell Line; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005144:F Factor; D005347:Fibroblasts; D011564:Furocoumarins; D017930:Genes, Reporter; D005819:Genetic Markers; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis; D008164:Luminescent Proteins / Q000096:biosynthesis; D008322:Mammals; D011107:Polylysine; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D014162:Transfection / Q000379:methods*
D011564:Furocoumarins; D005819:Genetic Markers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011107:Polylysine; D008156:Luciferases