title: Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells.
abstract: The ability to produce extremely small and circular supercoiled vectors has opened new territory for improving non-viral gene therapy vectors. In this work, we compared transfection of supercoiled DNA vectors ranging from 383 to 4,548 bp, each encoding shRNA against GFP under control of the H1 promoter. We assessed knockdown of GFP by electroporation into HeLa cells. All of our vectors entered cells in comparable numbers when electroporated with equal moles of DNA. Despite similar cell entry, we found length-dependent differences in how efficiently the vectors knocked down GFP. As vector length increased up to 1,869 bp, GFP knockdown efficiency per mole of transfected DNA increased. From 1,869 to 4,257 bp, GFP knockdown efficiency per mole was steady, then decreased with increasing vector length. In comparing GFP knockdown with equal masses of vectors, we found that the shorter vectors transfect more efficiently per nanogram of DNA transfected. Our results rule out cell entry and DNA mass as determining factors for gene knockdown efficiency via electroporation. The length-dependent effects we have uncovered are likely explained by differences in nuclear translocation or transcription. These data add an important step towards clinical applications of non-viral vector delivery.
authors.affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, United States of America.
authors.forename: Benjamin D
authors.initials: BD
authors.lastname: Hornstein
authors.affiliation: Post-Baccaleureate Research Education Program, Baylor College of Medicine, Houston, TX, United States of America.
authors.forename: Dany
authors.initials: D
authors.lastname: Roman
authors.affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, United States of America.|Verna and Marrs Mclean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, United States of America.|Department of Pharmacology, Baylor College of Medicine, Houston, TX, United States of America.
authors.forename: Lirio M
authors.initials: LM
authors.lastname: Arévalo-Soliz
authors.affiliation: Department of Pathology, Texas Children's Hospital, Houston, TX, United States of America.
authors.forename: Melinda A
authors.initials: MA
authors.lastname: Engevik
authors.affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, United States of America.|Verna and Marrs Mclean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, United States of America.|Department of Pharmacology, Baylor College of Medicine, Houston, TX, United States of America.
authors.forename: Lynn
authors.identifier: http://orcid.org/0000-0003-0533-1637
authors.initials: L
authors.lastname: Zechiedrich
mesh_terms: D045744:Cell Line, Tumor; D004270:DNA, Circular / Q000235:genetics*; D004278:DNA, Superhelical / Q000235:genetics; D018274:Electroporation / Q000379:methods; D055785:Gene Knockdown Techniques / Q000379:methods; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection / Q000379:methods
chemical_list: D004270:DNA, Circular; D004278:DNA, Superhelical; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins