Abstract:
The ability to introduce DNA and express custom DNA sequences in bacteria
opened the door for improvements in a large number of fields including agriculture,
pharmacology, medicine, nutrition, etc. The ability to introduce foreign DNA sequences
into mammalian cells in an efficient manner would have a large impact on therapeutic
applications especially gene therapy. The methods in use today suffer from low
efficiencies and sometimes toxicity. In this work a number of factors were evaluated for
their effect onONA uptake efficiency.
The factors studied included exposure to sublethal concentration of hydrogen
peroxide which have been show to lead to destabilisation ofthe lysosomes. These
exposures have proven to be very toxic to cells when combined with either the calcium
phosphate or the lipofectAMINE® transfection methods. Another factor evaluated was
exposure to Electro-Magnetic Fields (EMF). This was fuelled by the fact that EMF have
been shown to mediate a number of effects on cell structure and/or physiology. EMF
exposure by itself was not sufficient to induce the cells to pick up the DNA, therefore its
effect on calcium phosphate and lipofectAMINE® was tested. Although some positive
results were obtained, the variability of these results exceeded by far any observed
enhancements which discouraged any further work on EMF. Also tested was the possible
effect the presence of the cytomegalovirus (CMV) sequence might have on DNA uptake
(based on previous results in this lab). It was found that the presence ofCMV in the DNA
sequence does not enhance uptake or slow down degradation of the internalised DNA.
The final factor tested was the effect of basic amino acids on transfection efficiency. It
was found that arginine can enhance DNA uptake by about 170% v/ith calcium
phosphate and about 200% with LipofectAMINE®. A model was proposed to explain
the effect of arginine as well as the lack of effect from other amino acids.
