Lentivirus-based gene delivery in mouse embryonic stem cells

Yoshikazu Kosaka, Naoya Kobayashi, Takuya Fukazawa, Toshinori Totsugawa, Masanobu Maruyama, Chen Yong, Takashi Arata, Hideaki Ikeda, Kazuya Kobayashi, Tadayoshi Ueda, Yuzuru Kurabayashi, Noriaki Tanaka

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

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. Materials and methods: Recombinant HFV-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.

Original languageEnglish (US)
Pages (from-to)271-277
Number of pages7
JournalArtificial Organs
Volume28
Issue number3
DOIs
StatePublished - Mar 2004
Externally publishedYes

Fingerprint

Lentivirus
Green Fluorescent Proteins
Stem cells
Genes
Proteins
Embryonic Stem Cells
Severe Combined Immunodeficiency
Teratoma
HIV-1
Gene transfer
Infection
Therapeutic Human Experimentation
Neomycin
Flow cytometry
Lac Operon
Cytotoxicity
Mouse Embryonic Stem Cells
Gene expression
Escherichia coli
Cell- and Tissue-Based Therapy

Keywords

  • Embryonic stem cell
  • Gene transfer
  • Lentivirus vector

ASJC Scopus subject areas

  • Biophysics

Cite this

Kosaka, Y., Kobayashi, N., Fukazawa, T., Totsugawa, T., Maruyama, M., Yong, C., ... Tanaka, N. (2004). Lentivirus-based gene delivery in mouse embryonic stem cells. Artificial Organs, 28(3), 271-277. https://doi.org/10.1111/j.1525-1594.2004.47297.x

Lentivirus-based gene delivery in mouse embryonic stem cells. / Kosaka, Yoshikazu; Kobayashi, Naoya; Fukazawa, Takuya; Totsugawa, Toshinori; Maruyama, Masanobu; Yong, Chen; Arata, Takashi; Ikeda, Hideaki; Kobayashi, Kazuya; Ueda, Tadayoshi; Kurabayashi, Yuzuru; Tanaka, Noriaki.

In: Artificial Organs, Vol. 28, No. 3, 03.2004, p. 271-277.

Research output: Contribution to journalArticle

Kosaka, Y, Kobayashi, N, Fukazawa, T, Totsugawa, T, Maruyama, M, Yong, C, Arata, T, Ikeda, H, Kobayashi, K, Ueda, T, Kurabayashi, Y & Tanaka, N 2004, 'Lentivirus-based gene delivery in mouse embryonic stem cells', Artificial Organs, vol. 28, no. 3, pp. 271-277. https://doi.org/10.1111/j.1525-1594.2004.47297.x
Kosaka Y, Kobayashi N, Fukazawa T, Totsugawa T, Maruyama M, Yong C et al. Lentivirus-based gene delivery in mouse embryonic stem cells. Artificial Organs. 2004 Mar;28(3):271-277. https://doi.org/10.1111/j.1525-1594.2004.47297.x
Kosaka, Yoshikazu ; Kobayashi, Naoya ; Fukazawa, Takuya ; Totsugawa, Toshinori ; Maruyama, Masanobu ; Yong, Chen ; Arata, Takashi ; Ikeda, Hideaki ; Kobayashi, Kazuya ; Ueda, Tadayoshi ; Kurabayashi, Yuzuru ; Tanaka, Noriaki. / Lentivirus-based gene delivery in mouse embryonic stem cells. In: Artificial Organs. 2004 ; Vol. 28, No. 3. pp. 271-277.
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abstract = "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. Materials and methods: Recombinant HFV-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.",
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AU - Maruyama, Masanobu

AU - Yong, Chen

AU - Arata, Takashi

AU - Ikeda, Hideaki

AU - Kobayashi, Kazuya

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