title: A New Broad Range Plasmid for DNA Delivery in Eukaryotic Cells Using Lactic Acid Bacteria: In Vitro and In Vivo Assays.
abstract: Lactococcus lactisis well documented as a promising candidate for development of novel oral live vaccines. It has been broadly engineered for heterologous expression, as well as for plasmid expression vector delivery, directly inside eukaryotic cells, for DNA vaccine, or as therapeutic vehicle. This work describes the characteristics of a new plasmid, pExu (extra chromosomal unit), for DNA delivery usingL. lactisand evaluates its functionality both by in vitro and in vivo assays. This plasmid exhibits the following features: (1) a theta origin of replication and (2) an expression cassette containing a multiple cloning site and a eukaryotic promoter, the cytomegalovirus (pCMV). The functionality of pExu:egfpwas evaluated by fluorescence microscopy. TheL. lactisMG1363 (pExu:egfp) strains were administered by gavage to Balb/C mice and the eGFP expression was monitored by fluorescence microscopy. The pExu vector has demonstrated an excellent stability either inL. lactisor inEscherichia coli. The eGFP expression at different times in in vitro assay showed that 15.8% of CHO cells were able to express the protein after transfection. The enterocytes of mice showed the expression of eGFP protein. Thus,L. lactiscarrying the pExu is a good candidate to deliver genes into eukaryotic cells.
authors.affiliation: Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (ICB/UFMG), 31270-901 Belo Horizonte, Brazil.
authors.forename: Pamela
authors.initials: P
authors.lastname: Mancha-Agresti
authors.affiliation: Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (ICB/UFMG), 31270-901 Belo Horizonte, Brazil; CEFET - Centro Federal de Educação Tecnológica de Minas Gerais, Coordenação de Ciências, Campus I, 30421-169 Belo Horizonte, Brazil.
authors.forename: Mariana Martins
authors.initials: MM
authors.lastname: Drumond
authors.affiliation: Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (ICB/UFMG), 31270-901 Belo Horizonte, Brazil.
authors.forename: Fillipe Luiz Rosa do
authors.initials: FL
authors.lastname: Carmo
authors.affiliation: Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (ICB/UFMG), 31270-901 Belo Horizonte, Brazil.
authors.forename: Monica Morais
authors.initials: MM
authors.lastname: Santos
authors.affiliation: Laboratório de Inovação Biotecnológica, Fundação Ezequiel Dias (FUNED), Belo Horizonte, 30510-010 Minas Gerais, Brazil.
authors.forename: Janete Soares Coelho Dos
authors.initials: JS
authors.lastname: Santos
authors.affiliation: School of Biosciences and Veterinary Medicine, University of Camerino, 62032 Camerino, Italy.
authors.forename: Franco
authors.initials: F
authors.lastname: Venanzi
authors.affiliation: Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Chatel
authors.affiliation: Laboratório de Inovação Biotecnológica, Fundação Ezequiel Dias (FUNED), Belo Horizonte, 30510-010 Minas Gerais, Brazil.
authors.forename: Sophie Yvette
authors.initials: SY
authors.lastname: Leclercq
authors.affiliation: Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (ICB/UFMG), 31270-901 Belo Horizonte, Brazil.
authors.forename: Vasco
authors.initials: V
authors.lastname: Azevedo
keywords: DNA mucosal delivering; Lactococcus lactis; lactic acid bacteria; shuttle plasmid