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Not content with using mass spectrometry (MS) to analyse biological material, Japanese scientists have now shown that it can also be used to modify biological material. Specifically, they have shown that the popular MS ionisation technique known as electrospray ionisation (ESI) can introduce genetic material into cells. In ESI, the effluent from a high performance liquid chromatography column is pushed through an electrically charged capillary tip, emerging as a charged aerosol spray that is then sent into a mass spectrometer. This process ionises the analytes in the effluent via the formation and evaporation of charged droplets. It is a fairly gentle ionisation technique, which doesn't tend to cause the analyte molecules to fragment, and so has become a popular method for ionising large macromolecules and biomolecules, such as proteins and oligonucleotides. More recently, the development of desorption ESI (DESI), in which charged water droplets are fired at the surface of a solid sample, has shown that electrospray technology can be extended to ionising intact samples under ambient conditions. This led two Japanese scientists, Kazuto Ikemoto from the Mitsubushi Gas Chemical Company and Takafumi Sakai from Saitama University, to speculate what else the technology might be able to do. In particular, they wondered whether it could introduce foreign material into cells, a process that is commonly known as transfection. 'Dr Ikemoto first had the idea that the electrospray method could be applied to the transfection of micro-organisms,' explains Sakai. 'I was using [conventional] transfection techniques for mammalian cell lines, but had become dissatisfied with the results and sought a more effective method. We met at the annual meeting of the Japanese Society of Applied Physics and started collaborating.' Numerous methods are available for introducing foreign material such as DNA into cells. But they either tend to damage many of the cells, as is the case with the gene guns that fire DNA-coated metal particles into cells, or can be fairly complicated to perform, as is the case with using viruses to inject DNA into cells. Ikemoto and Sakai were interested to know whether electrospray technology could form the basis for a gentler version of another transfection method known as electroporation. This involves opening up pores in cell membranes by applying an electric current, thereby allowing foreign material outside of the cells to flow inside. In the electrospray version, these pores would be produced by firing charged water droplets at the cells. Testing this method on Chinese hamster ovary cells growing in a culture dish that contained DNA coding for a green fluorescent protein, Ikemoto and Sakai first discovered that, compared to ESI, they needed to apply a much higher voltage to produce the charged droplets (7-18kV rather than 1-3kV). This is because they were conducting electrospray using purified water, which has a higher surface tension than the toxic organic solvents usually used with ESI. Nevertheless, this test also confirmed that the electrospray method was able to introduce foreign DNA into the ovary cells without damaging them, with around 1% of the cells subsequently displaying a green fluorescent glow. This transfection rate could be increased by raising the applied voltage and ensuring that the cells were swelled with water. The scientists also found that the rate was much higher when the DNA was added to the culture before electrospraying rather than when it was added directly after. This implies that the holes produced by the charged water particles only persist for around one minute before closing up. Ikemoto and Sakai then went on to show that this electrospray method could also successfully introduce DNA into chicken embryos and the bacterium Escherichia coli. They are now looking at ways to improve the transfection rate still further and have already developed a computer-controlled pedestal that allows the electrospray to be applied over a greater area of the culture dish. Related links:
The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd. |
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