Title: Recombinant adeno-associated virus serotype 9 transfection of rat cardiac fibroblasts in vitro
Abstract: Objective:To assess the transfection efficiency of recombinant adeno-associated virus serotype 9 mediated enhanced green fluorescent protein(rAAV9-EGFP) to rat cardiac fibroblasts and the impact on growth of rat cardiac fibroblasts.Methods:The rat cardiac fibroblasts were cultured and dissociated.The rAAV9-EGFP was transfected into rat cardiac fibroblasts at different multiplicities of infection(MOI=1×104,1×105,1×106).EGFP expression in the cells was observed under inverted fluorescence microscope,and the EGFP-positive cell percentage was determined by flow cytometry.AlamarBlue assay was used to assess the proliferation of the transfected cells.Results:The cells with rAAV9-EGFP transfection at MOI of 1×106 began to exhibit EGFP expression 24 h after transfection and the cells transfection at MOI of 1×105 and 1×104 began to exhibit EGFP expression 48 h after transfection.The cells growth was normal in all three groups under inverted fluorescence microscope.EGFP expression reached the maximum at 120 h,at the point of which the transduction efficiency of rAAV9-EGFP in rat cardiac fibroblasts cells was(2.6±0.2)%,(7.3±1.4)% and(45.1±2.7)% corresponding to MOIs of 1×104,1×105 and 1 ×106,respectively.The fluorescence intensity increased with the MOI and time increasing.AlamarBlue assay did not reveal significant difference in the absorbance between the transfected cells and the control cells.Conclusion:rAAV9-EGFP gene can be stably and efficiently expressed in rat cardiac fibroblasts without inhibiting cell growth,suggesting the potential of rAAV9 as a safe and efficient vector for gene therapy of heart disease research.
Publication Year: 2010
Publication Date: 2010-01-01
Language: en
Type: article
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Cited By Count: 1
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