Green fluorescent protein as indicator of nonviral transient transfection efficiency in endometrial and testicular biopsies.
In the last years, physical and chemical methods of plasmid delivery have revolutionized the efficiency of nonviral gene transfer, and the success of gene therapy is largely dependent upon the development of gene-delivery methods. The nonviral techniques that lead to a direct transfer of DNA into tissue fragments, like electroporation (EP) and lipofection delivery systems are still insufficiently investigated. Our aim was to test the efficiency of EP and lipofection protocols in endometrial and testicular tissue fragments, using a naked plasmid DNA encoding green fluorescent protein (GFP). Because the transfection efficiency depends upon several factors, we tried to optimize the transfection conditions by testing different lipofectamine 2000 and plasmid ratios, electrical parameters, and culture after transfection. Our results show that these two nonviral methods of gene delivery are feasible and efficient in gene transfection of endometrial and testicular tissue biopsies. We found that the most performing ratio of plasmid:lipofectamine was 10:50 for transient lipofection, whereas two pulses for 10 s at 960 microF of capacitance, 200 V of voltage were the most favorable electrical parameters for EP efficiency in the presence of 5 microL of phMGFP plasmid. After lipofection and EP, the highest GFP intensity was observed respectively after 48 and 72 h of tissue fragment culturing. In conclusion, nonviral methods are attractive for an improvement of the gene therapy and our protocol could provide useful indications for in vivo gene therapy applications.
Department of Molecular Pathology and Innovative Therapies-Histology, Marche Polytechnic University, Ancona, Italy.
Antonio
A
Zizzi
Daniele
D
Minardi
Andrea
A
Ciavattini
Federica
F
Giantomassi
Rodolfo
R
Montironi
Giovanni
G
Muzzonigro
Roberto
R
Di Primio
Guendalina
G
Lucarini
D001706:Biopsy; D004717:Endometrium; D005260:Female; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008055:Lipids; D008297:Male; D008856:Microscopy, Fluorescence / Q000379:methods; D010957:Plasmids; D013194:Staining and Labeling / Q000379:methods; D013737:Testis; D014162:Transfection
D008055:Lipids; C086724:Lipofectamine; D049452:Green Fluorescent Proteins