Highly efficient cell-mediated gene transfer using non-viral vectors and FuGene (TM) 6: in vitro and in vivo studies

Citation
I. Hellgren et al., Highly efficient cell-mediated gene transfer using non-viral vectors and FuGene (TM) 6: in vitro and in vivo studies, CELL MOL L, 57(8-9), 2000, pp. 1326-1333
Citations number
27
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Cell & Developmental Biology
Journal title
CELLULAR AND MOLECULAR LIFE SCIENCES
ISSN journal
1420-682X → ACNP
Volume
57
Issue
8-9
Year of publication
2000
Pages
1326 - 1333
Database
ISI
SICI code
1420-682X(200008)57:8-9<1326:HECGTU>2.0.ZU;2-H
Abstract
The present study was undertaken to develop an efficient non-viral gene del ivery system for cardiovascular gene therapy. We investigated transfection efficiency and toxic properties of the new transfection reagent, FuGene(TM) 6, and compared it with two other transfection reagents, Tfx(TM)-50 and Lip oTaxi(TM). For in vivo experiments, the plasmid was delivered intramuscular ly via transplantation of fibroblasts transfected with plasmid and FuGene(T M)6. Conditions for efficient gene delivery were initially studied in vitro . Human and rabbit fibroblasts were isolated from skin, cultured and transf ected with phVEGF165 or pCMV beta gal plasmids, coding for vascular endothe lial growth factor (VEGF) or beta-galactosidase, respectively. The effect o f the DNA amount and the DNA:transfection reagent ratio on plasmid uptake w ere studied. Of the transfection reagents tested, only FuGene(TM)6 provided high-efficiency and dose-dependent plasmid transfer both for cell-localise d (beta-galactosidase) and secreted (VEGF) gene products. When analysed wit h an MTT assay, FuGene(TM)6 showed no toxicity at low doses. Optimised cond itions were applied for in vivo reporter gene delivery. Rabbits were inject ed intramuscularly with ex vivo-transfected fibroblasts. As in in vitro stu dies, ex vivo-transfected fibroblasts showed highly efficient gene expressi on in vivo. Tissue sections were analysed with macrophage-specific immunost aining. No signs of inflammation were seen in the region of fibroblast inje ction. This study demonstrates that FuGenc(TM)6 is a highly efficient trans fection reagent that may be useful for in vitro son-viral transfection of p rimary human and rabbit fibroblasts and for in vivo therapeutic non-viral g ene delivery.