SV40 DNA transfection of cells in suspension: analysis of efficiency of transcription and translation of T-antigen

A modification of the Graham and Van der Eb (1974) DNA-calcium phosphate coprecipitation technique is shown to routinely transfect 15% of CV-1 cells with SV40 DNA. The transfection is done in suspension after detachment of cells by trypsin digestion. Transfection efficiency was measured by staining cells for the presence of SV40 T-antigen by indirect immunofluorescence and by assaying for the presence of SV40 early message by the Berk and Sharp (1978) technique.     

High performance DNA nano-carriers of carbonate apatite: multiple factors in regulation of particle synthesis and transfection efficiency

Increasing attention is being paid on synthetic DNA delivery systems considering some potential life-threatening effects of viral particles, for development of gene-based nanomedicine in the 21st century. In the current nonviral approaches, most of the efforts have been engaged with organic macromolecules like lipids, polymers, and peptides, but comparatively fewer attempts were made to evaluate the potential of inorganic materials for gene delivery. We recently reported that biodegradable nanoparticles of carbonate apatite are highly efficient in transfecting a wide variety of mammalian cells. Here we show that a number of parameters actively regulate synthesis of the nanoparticles and their subsequent transfection efficacy. Development of "supersaturation", which is the prerequisite for generation of such particles, could be easily modulated by reactant concentrations, pH of the buffered solution, and incubation temperatures, enabling us to establish a flexible particle generation process for highly productive trans-gene delivery. Carbonate incorporation into the particles have been proposed for generating nano-size particles resulting in cellular uptake of huge amount of plasmid DNA as well as endosome destabilization facilitating significant release of DNA from the endosomes.     

Linear DNA low efficiency transfection by liposome can be improved by the use of cationic lipid as charge neutralizer

A plasmid expressing the beta-galactosidase enzyme was used to transfect Vero cells in order to evaluate the efficiency of a liposome-mediated transfection by circular and linear DNA. The results obtained showed a low rate of transfection by linear DNA:liposome complexes. To explore whether the structure of the complexes was interfering with the transfection, atomic force microscopy (AFM) was used. It has confirmed the difference between the linear and circular condensates: whereas the circular DNA:liposome complexes presented compact spherical or cylindrical structures of about 100-800 nm, the linear DNA showed pearl necklace-like structures, with pearls varying from 250 to 400 nm. On the basis of the theory proposed by Kuhn et al. (1999), low concentrations of cationic amphihile were used to neutralize or reverse the DNA charge in order to improve the transfection efficiency of the linear DNA. Using this method, we were able to obtain the expression of the transgene without an associated toxicity observed with the linear DNA liposome delivery.