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Efficiency of the Elongation Factor-1α Promoter in Mammalian Embryonic Stem Cells Using Lentiviral Gene Delivery Systems

2007; Mary Ann Liebert, Inc.; Volume: 16; Issue: 4 Linguagem: Inglês

10.1089/scd.2006.0088

1557-8534

Sinae Kim, Gi Jin Kim, Hiroyuki Miyoshi, Sung‐Hwan Moon, Seong Eun Ahn, Jae Ho Lee, Hey Jin Lee, Kwang‐Yul Cha, Hyung‐Min Chung,

RNA Interference and Gene Delivery

The establishment of new technology for genetic modification in human embryonic stem (ES) cell lines has raised great hopes for achieving new ground in basic and clinical research. Recently, lentiviral vector technology has been shown to be highly effective and therefore could emerge as a popular tool for human ES cell genetic modification. The objectives of this study were to evaluate the efficiency of promoters in lentiviral gene delivery systems in mammalian ES cells, including mouse, monkey, and human, and to construct efficient and optimized conditions for lentivirus-mediated transfection systems. Mammalian ES cells were transfected with self-inactivating (SIN) human immunodeficiency virus type-1 (HIV-1)-based lentiviral vectors containing the human polypeptide chain elongation factor-1α (EF-1α) promoter or cytomegalovirus (CMV) promoter and analyzed by fluorescence-activated cell sorting (FACS) analysis for the expression of the enhanced green fluorescent protein (eGFP) reporter gene. The efficiency of the EF-1α promoter was higher than that of the CMV promoter in all ES cells tested. The EF-1α promoter efficiently drove gene expression (14.74%) compared with CMV promoter (3.69%) in human ES cells. We generated a stable eGFP+ human ES cell line (CHA3-EGFP human ES cells) that continuously expressed high levels of EGFP (∼95%) from the EF-1α promoter and was maintained for up to 60 weeks with undifferentiated proliferation. The established CHA3-EGFP human ES cell lines were characterized as being negative for nondifferentiation markers and teratoma formation. These results imply that genetic modification by lentiviral vectors with specific promoters in ES cells constitute a powerful tool for guided differentiation as well as gene therapy.