A method to monitor DNA transfer during transfection
Journal Title: The AAPS Journal - Year 1999, Vol 1, Issue 2
Abstract
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.
Authors and Affiliations
Andrew L. Johnson, Joseph A. Jurcisek, O. Joseph Trask, Jessie L. -S. Au
Keywords
Related Articles
- EP ID EP682111
- DOI 10.1208/ps010206
- Views 79
- Downloads 0
Engineered Mannitol Ternary Additives Improve Dispersion of Lactose–Salbutamol Sulphate Dry Powder Inhalations
The online version of this article (doi:10.1208/s12248-013-9476-4) contains supplementary material, which is available to authorized users.
An Exact Procedure for the Evaluation of Reference-Scaled Average Bioequivalence
The online version of this article (doi:10.1208/s12248-016-9873-6) contains supplementary material, which is available to authorized users.
Pharmacokinetics and Pharmacodynamics of Phenethyl Isothiocyanate: Implications in Breast Cancer Prevention
Phenethyl isothiocyanate (PEITC)—a naturally occurring isothiocyanate in cruciferous vegetables—has been extensively studied as a chemopreventive agent in several preclinical species and in humans. Pharma...
Genetic factors in the predisposition to drug-induced hypersensitivity reactions
Drug hypersensitivity reactions can occur with most drugs, although the frequency, severity, and clinical manifestations vary. Case reports have suggested that there may be familial clustering of drug hypersensitivity su...
Utility of Physiologically Based Absorption Modeling in Implementing Quality by Design in Drug Development
The online version of this article (doi:10.1208/s12248-010-9250-9) contains supplementary material, which is available to authorized users.