title: Confocal laser microscopy of chondrocytes that received gene transfer using in vitro electroporation.
abstract: To develop a gene therapy for osteopathies, this study was conducted to establish a method of transferring the BMP gene, a bone formation factor, to cells and administering the cells with BMP expression to patients with osteopathies. Although virus vectors are frequently used for gene transfer, there has been reported a death case of gene therapy using the adenovirus vector. Therefore, various efforts have been made to prevent such complications. In the present study, we used electroporation by which gene transfer can be efficiently performed without inducing severe complications after electric perforation of the cell membrane. Human bone tissues were initially collected intraoperatively, and BMP-2 and Smad4 genes were cloned and integrated into GFP and DsRed plasmid vectors. Using in vitro electroporation, these plasmid vectors were transferred to the cultured chondrocytes (KTN-1) derived from human herniated intervertebral disk. Confocal laser microscopy revealed that the BMP gene was successfully transferred to the nucleus of chondrocytes in the presence of Smad. Since electroporation facilitated human gene transfer to the target cells, gene therapy using electroporation may facilitate individualized treatment for patients.
authors.affiliation: Department of Orthopaedic Surgery, School of Medicine, Kurume University, Kurume 830-0011, Japan. michiyo@med.kurume-u.ac.jp
authors.forename: Michiyo
authors.initials: M
authors.lastname: Tsuru
authors.forename: Kensei
authors.initials: K
authors.lastname: Nagata
authors.forename: Takato
authors.initials: T
authors.lastname: Ueno
authors.forename: Atsuo
authors.initials: A
authors.lastname: Jimi
authors.forename: Shinshi
authors.initials: S
authors.lastname: Noda
authors.forename: Shizuka
authors.initials: S
authors.lastname: Iida
authors.forename: Michio
authors.initials: M
authors.lastname: Sata
mesh_terms: D001483:Base Sequence; D001847:Bone Diseases / Q000628:therapy; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000235:genetics; D019902:Chondrocytes / Q000648:ultrastructure*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics; D018274:Electroporation / Q000379:methods*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D006801:Humans; D066298:In Vitro Techniques; D007834:Lasers; D018613:Microscopy, Confocal / Q000379:methods*; D051901:Smad4 Protein; D015534:Trans-Activators / Q000235:genetics; D016212:Transforming Growth Factor beta
chemical_list: C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; C497796:SMAD4 protein, human; D051901:Smad4 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta