Use of flow cytometry to rapidly optimize the transfection of animal cells.
Plasmid transfection is the first step in the generation of stably transformed animal cells and is also a useful tool for analyzing transient gene expression. Maximizing the transfection efficiency and expression level from the introduced plasmid is critical to the success of these processes. By means of lipid-mediated transfection, a plasmid vector expressing the green fluorescence reporter protein has been coupled with flow cytometry to conveniently investigate those parameters that impact the efficacy of transfection of lepidopteran insect cells. The key feature of this technique is the rapid and simultaneous quantification of transfection efficiency and heterologous protein expression level per cell. Using this technique, we developed an optimized transfection protocol for insect cells by investigating the following parameters: lipid incubation time, lipid/DNA mixture incubation time, lipid and DNA concentration, incubation vessel and transfection duration. Following optimization, transfection efficiencies of 37%-40% were obtained for Bombyx mori Bm5 and Spodoptera frugiperda Sf-21 cells.
Keith
M B
MB
University of Calgary, AB, Canada.
Farrell
P J
PJ
Iatrou
K
K
Behie
L A
LA
D000818:Animals; D012831:Bombyx / Q000235:genetics; D002470:Cell Survival; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D007915:Lepidoptera; D050356:Lipid Metabolism; D008164:Luminescent Proteins / Q000235:genetics; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010957:Plasmids; D018411:Spodoptera / Q000235:genetics; D013997:Time Factors; D014162:Transfection; D014170:Transformation, Genetic
D008164:Luminescent Proteins; D010714:Phosphatidylethanolamines; D049452:Green Fluorescent Proteins; C020888:1,2-dielaidoylphosphatidylethanolamine; D004247:DNA