Lentivirus-based gene delivery in mouse embryonic stem cells.
BACKGROUND
Embryonic stem (ES) cells are widely used in therapeutic research as an unlimited source of cell therapy. Therefore, it is of great value to find a way to efficiently manipulate ES cells. HIV-1-derived lentiviral vectors are now considered to be an efficient vehicle for delivering genes into a variety of cells. In this study, we examined the efficacy of lentivirus-based gene delivery into mouse ES (mES) cells.


METHODS
Recombinant HIV-I-based lentiviral vectors Lt-GFP, expressing green fluorescent protein (GFP), and Lt-LacZ, expressing E. coli LacZ gene in conjunction with neomycin resistance gene, were generated using a FuGENE 6 transduction method and used for transducing ES cells derived from 129Sv mice. Lentiviral transduction efficacy was evaluated by GFP expression assay using flow cytometry and by X-gal staining. The in vivo potential of developing teratoma of such transduced mES cells was examined in severe combined immunodeficiency (SCID) mice.


RESULTS
FuGENE 6 showed no considerable transduction-associated cytotoxicity. The expression rate of GFP and LacZ of mES cells increased on a multiplicity of infection (MOI)-dependent manner with the amount of Lt-GFP and Lt-LacZ used. Approximately 42% of mES cells were positive for GFP after infection of Lt-GFP at an MOI of 30. Notably, after G418 selection, nearly 100% of Lt-LacZ-transduced mES cells were positive for LacZ and formed teratomas in SCID mice.


CONCLUSIONS
This work demonstrates that HIV-I-based lentiviral vectors are capable of transducing mES cells. Lentiviral vectors may facilitate an advance in the field of gene transfer and expression in various types of ES cells, including human ES cells.
Kosaka
Yoshikazu
Y
Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan.
Kobayashi
Naoya
N
Fukazawa
Takuya
T
Totsugawa
Toshinori
T
Maruyama
Masanobu
M
Yong
Chen
C
Arata
Takashi
T
Ikeda
Hideaki
H
Kobayashi
Kazuya
K
Ueda
Tadayoshi
T
Kurabayashi
Yuzuru
Y
Tanaka
Noriaki
N
D000818:Animals; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007763:Lac Operon / Q000235:genetics; D016086:Lentivirus; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D013234:Stem Cells; D013724:Teratoma / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods*; D016896:Treatment Outcome
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins