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A simple and efficient purification of transduced cells by using green fluorescent protein gene as a selection marker
Author(s) -
Shimizu Takashi,
Ando Kiyoshi,
Kimura Minoru,
Miyatake Hiroko,
Inokuchi Sadaki,
Takakura Iwao,
Migita Makoto,
Shimada Takashi,
Kato Shunichi
Publication year - 1998
Publication title -
pediatrics international
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.49
H-Index - 63
eISSN - 1442-200X
pISSN - 1328-8067
DOI - 10.1111/j.1442-200x.1998.tb01995.x
Subject(s) - green fluorescent protein , retrovirus , transfection , genetic enhancement , viral vector , recombinant dna , microbiology and biotechnology , flow cytometry , cell culture , virus , gene , virology , medicine , biology , biochemistry , genetics
Background: Simple and efficient method for the selection of transduced cells would greatly facilitate the clinical utilization of retrovirus vectors. We developed a therapeutic bicistronic retrovirus vector for Gaucher disease, MFG‐GC‐GFP, which contains the human glucocerebrosidase (GC) gene and the green fluorescent protein (GFP) gene of the jellyfish Aequorea victoria as a vital selection marker, and investigated its applicability as gene therapy for Gaucher disease.Methods and Results: A packaging cell line, GP + envAM12, was transfected with MFG‐GC‐GFP and, thus, produced a high titer recombinant virus (1.0 × 10 6 c.f.u./mL) in the culture supernatant. The expression level of GFP was correlated with the virus production in cells. The recombinant virus infected skin fibroblasts from a Gaucher patient and a sorted fraction of the cells expressing GFP by flow cytometry exhibited almost a six‐fold higher activity of GC than normal fibroblasts.Conclusions: These data indicate that MFG‐GC‐GFP enables the one‐step purification of a transduced fraction of target cells and is, therefore, considered to be a useful therapeutic vector for the experimental gene therapy of Gaucher disease.