The journal of gene medicine · 2004
Efficient transfection of non-proliferating human airway epithelial cells with a synthetic vector system.
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Meng Qing-Hai, Robinson Danielle, Jenkins R Gisli, McAnulty Robin J, Hart Stephen L
Аннотация
BACKGROUND: The poor transfection efficiency of most synthetic vectors in non-dividing, airway epithelial cells is limiting the development of gene therapy for respiratory diseases such as cystic fibrosis.
METHODS: Cultured airway epithelial cells, quiescent or replicating, were transfected with a lipid/peptide synthetic vector system and transfection efficiency assessed by luciferase assays or flow cytometry for green fluorescent protein expression. Tight junctions were disrupted by treatment with ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA). Cell division was inhibited by aphidicolin and assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation and assay by fluorescence microscopy or flow cytometry. In vivo transfection was performed by intratracheal instillation.
RESULTS: Initial studies showed that this synthetic vector transfected 8% of non-dividing epithelial cells in culture compared with 45% of subconfluent, dividing cells. However, formulation of the vector with EGTA, which disrupts cellular tight junctions, induced a four-fold increase in the transfection efficiency of confluent, non-mitotic cells. The EGTA enhancement of transfection also correlated with a four-fold increase in vector binding to the cells, presumably via exposed basolateral receptors. Flow cytometry and double immunofluorescent staining for reporter gene expression and BrdU incorporation indicated efficient transfection of non-proliferating cells in a confluent monolayer by this vector. Furthermore, in vivo transfection of murine lung by intratracheal administration showed a six-fold enhancement of luciferase expression when lungs were pre-treated with EGTA.
CONCLUSIONS: This study confirms the hypothesis that a lipid/peptide vector transfects non-dividing cells with high efficiency which contributes to its previously reported efficiency in transfecting bronchial epithelium, making it a good choice for gene transfer studies in the lung.
METHODS: Cultured airway epithelial cells, quiescent or replicating, were transfected with a lipid/peptide synthetic vector system and transfection efficiency assessed by luciferase assays or flow cytometry for green fluorescent protein expression. Tight junctions were disrupted by treatment with ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA). Cell division was inhibited by aphidicolin and assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation and assay by fluorescence microscopy or flow cytometry. In vivo transfection was performed by intratracheal instillation.
RESULTS: Initial studies showed that this synthetic vector transfected 8% of non-dividing epithelial cells in culture compared with 45% of subconfluent, dividing cells. However, formulation of the vector with EGTA, which disrupts cellular tight junctions, induced a four-fold increase in the transfection efficiency of confluent, non-mitotic cells. The EGTA enhancement of transfection also correlated with a four-fold increase in vector binding to the cells, presumably via exposed basolateral receptors. Flow cytometry and double immunofluorescent staining for reporter gene expression and BrdU incorporation indicated efficient transfection of non-proliferating cells in a confluent monolayer by this vector. Furthermore, in vivo transfection of murine lung by intratracheal administration showed a six-fold enhancement of luciferase expression when lungs were pre-treated with EGTA.
CONCLUSIONS: This study confirms the hypothesis that a lipid/peptide vector transfects non-dividing cells with high efficiency which contributes to its previously reported efficiency in transfecting bronchial epithelium, making it a good choice for gene transfer studies in the lung.