title: Sequence-Modified Antibiotic Resistance Genes Provide Sustained Plasmid-Mediated Transgene Expression in Mammals.
abstract: Conventional plasmid vectors are incapable of achieving sustained levels of transgene expression in vivo even in quiescent mammalian tissues because the transgene expression cassette is silenced. Transcriptional silencing results from the presence of the bacterial plasmid backbone or virtually any DNA sequence of >1 kb in length placed outside of the expression cassette. Here, we show that transcriptional silencing can be substantially forestalled by increasing the An/Tn sequence composition in the plasmid bacterial backbone. Increasing numbers of An/Tn sequences increased sustained transcription of both backbone sequences and adjacent expression cassettes. In order to recapitulate these expression profiles in compact and portable plasmid DNA backbones, we engineered the standard kanamycin or ampicillin antibiotic resistance genes, optimizing the number of An/Tn sequence without altering the encoded amino acids. The resulting vector backbones yield sustained transgene expression from mouse liver, providing generic DNA vectors capable of sustained transgene expression without additional genes or mammalian regulatory elements.
authors.affiliation: Department of Pediatrics, Stanford University School of Medicine, Palo Alto, CA 94305, USA; Department of Genetics, Stanford University School of Medicine, Palo Alto, CA 94305, USA.
authors.forename: Jiamiao
authors.initials: J
authors.lastname: Lu
authors.affiliation: Department of Pediatrics, Stanford University School of Medicine, Palo Alto, CA 94305, USA; Department of Genetics, Stanford University School of Medicine, Palo Alto, CA 94305, USA.
authors.forename: Feijie
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Department of Genetics, Stanford University School of Medicine, Palo Alto, CA 94305, USA; Department of Pathology, Stanford University School of Medicine, Palo Alto, CA 94305, USA.
authors.forename: Andrew Z
authors.initials: AZ
authors.lastname: Fire
authors.affiliation: Department of Pediatrics, Stanford University School of Medicine, Palo Alto, CA 94305, USA; Department of Genetics, Stanford University School of Medicine, Palo Alto, CA 94305, USA. Electronic address: markay@stanford.edu.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Kay
mesh_terms: D000667:Ampicillin / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics*; D005260:Female; D020868:Gene Silencing; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007612:Kanamycin / Q000494:pharmacology; D008099:Liver / Q000378:metabolism / Q000821:virology; D051379:Mice; D008810:Mice, Inbred C57BL; D059372:Nucleotide Motifs; D010957:Plasmids / Q000737:chemistry* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects; D052736:Rous sarcoma virus / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D019076:Transgenes; D000515:alpha 1-Antitrypsin / Q000235:genetics* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D000515:alpha 1-Antitrypsin; D007612:Kanamycin; D000667:Ampicillin
keywords: An/Tn tracts; antibiotic resistance gene; non-coding transcription; nucleosomes; plasmid silencing; plasmid vector