A new tool for efficient transfection of dog and human thyrocytes in primary culture.
The introduction of exogenous DNA into mammalian cells is commonly used to study the functions of gene products. However cells in primary culture are usually refractory to most transfection systems. Here we investigated the ability of a new lipid formulation, FuGENE 6 transfection reagent, to promote DNA uptake into dog and human thyroid cells in primary culture. Gene transfer was monitored by the expression of a Green Fluorescent Protein (GFP) reporter gene. We report that FuGENE 6 is particularly suited for the transfection of thyroid cells and does not interfere with their normal growth. Optimization of the experimental conditions, such as DNA amount, DNA/lipid ratio, cell density and incubation with the transfection mixture, was achieved by evaluating the percentage of GFP-expressing cells by FACS analysis. FuGENE 6 allowed us to obtain 8-15% thyrocytes expressing the reporter gene which represents an efficiency 100-fold superior to other transfection methods.
Interdisciplinary Research Institute, Free University of Brussels, Belgium.
N
N
Uyttersprot
S
S
Costagliola
F
F
Miot
D000818:Animals; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004285:Dogs; D005434:Flow Cytometry; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents; D050356:Lipid Metabolism; D008164:Luminescent Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D013961:Thyroid Gland / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004247:DNA