title: Efficient gene expression in megakaryocytic cell line using nucleofection.
abstract: To clarify the mechanism of platelet production from megakaryocytes, expression of target proteins by gene transfection was examined using various gene delivery techniques. Transfection into hematopoietic cells, including megakaryocytes, by conventional gene delivery techniques such as electroporation and lipofection are known to be difficult. In this study, in addition to electroporation and lipofection, we tested other gene-transfer methods (nucleofection, transfection using inactivated virus envelope, and transferrin-linked cationic polymer) with the green fluorescent protein (GFP) gene into the human megakaryocytic cell line MEG-01. We found that nucleofection, which uses a combination of special electrical parameters and specific solutions, was the best, judging from the expression ratio of GFP-positive cells (approximately 70% of cells) and low toxicity. The efficiency of GFP expression was not related to the amount of pDNA delivered into the MEG-01 cells. To verify the utility of nucleofection, the thrombopoietin (TPO) receptor c-mpl was transfected into MEG-01 cells. Transfected cells showed a higher responsiveness to TPO than mock-transfected MEG-01 cells. We propose that nucleofection is a useful method for transfecting target genes to megakaryocytic cells when addressing the mechanism of platelet production.
authors.affiliation: Department of Pharmacokinetics and Biopharmaceutics, Subdivision of Biopharmaceutical Sciences, Institute of Health Biosciences, The University of Tokushima, 1-78-1, Sho-machi, Tokushima 770-8505, Japan. y_isakari@research.otsuka.co.jp
authors.forename: Yoshimasa
authors.initials: Y
authors.lastname: Isakari
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Harada
authors.forename: Dai
authors.initials: D
authors.lastname: Ishikawa
authors.forename: Kuniko
authors.initials: K
authors.lastname: Matsumura-Takeda
authors.forename: Shinji
authors.initials: S
authors.lastname: Sogo
authors.forename: Tatsuhiro
authors.initials: T
authors.lastname: Ishida
authors.forename: Takao
authors.initials: T
authors.lastname: Taki
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kiwada
mesh_terms: D002460:Cell Line; D018274:Electroporation; D005822:Genetic Vectors; D006801:Humans; D008081:Liposomes; D008533:Megakaryocytes / Q000378:metabolism*; D053628:Receptors, Thrombopoietin / Q000235:genetics*; D029082:Sendai virus / Q000235:genetics; D014162:Transfection / Q000379:methods*
chemical_list: D008081:Liposomes; D053628:Receptors, Thrombopoietin; C082218:MPL protein, human