A method to monitor DNA transfer during transfection.
This report describes a method for monitoring the transfer of DNA during transfection. This method involves random labeling of plasmid DNA with fluorescein-12-dUTP, flow cytometric detection and sorting of the fluorescent transfected cells, and laser confocal fluorescence microscopic determination of the intracellular location of the plasmid DNA. By this method, >95% of the sorted cells were labeled, indicating a >95% specificity of the sorting procedure. The sorted cells were viable, as indicated by their ability to exclude trypan blue dye (>98% cells excluded the dye) and to maintain cell growth. The results of the kinetics of the Lipofectin transfection technology show that the fraction of the cells that internalized plasmid DNA increased from 10% at 1 hr after initiation of the transfection procedures to 18% at 3 hr. This method does not require protein expression, does not require the use of selection pressure such as drug treatment to isolate the cells that internalized DNA, and can be used to study the early events of DNA transfection.
Johnson
A L
AL
College of Pharmacy, The Ohio State University, 500 West 12th Aveneue, Columbus, OH 43210, USA.
Jurcisek
J A
JA
Trask
O J
OJ
Au
J L
JL
D002470:Cell Survival; D003856:Deoxyuracil Nucleotides; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010714:Phosphatidylethanolamines; D010957:Plasmids / Q000235:genetics*; D014162:Transfection / Q000379:methods; D014407:Tumor Cells, Cultured
D003856:Deoxyuracil Nucleotides; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008081:Liposomes; D010714:Phosphatidylethanolamines; C069632:fluorescein-11-deoxyuridine triphosphate; C020888:1,2-dielaidoylphosphatidylethanolamine