18 April 2011

Honing doors in cells

Cell transfection is an important tool for studying and elucidating the basic cellular processes and genotypic changes inside cells. One method to do so is optically assisted transfection, a process in which a highly localized laser beam alters the permeability of the plasma membrane, thereby allowing the entry of extracellular nucleic acids into a cell. Compared to other established transfection methods, it opens up the possibility to permeate the lipid cell membrane in a sterile and relatively non-invasive manner, especially in the field of drug delivery and gene therapy. The physical mechanisms for transfection by different laser types differ. In particular, femtosecond laser irradiation provides a popular and powerful method with strong applicability to single cell transfection. Yet, the efficiency of transfection still requires enhancement.

Optical transfection creates a transient permeability of the cell membrane so that plasmid DNA can enter the cytoplasm. However, in order for the plasmid to be transcribed it has to enter the nucleus of the cell, which can only take place during the mitotic division when the nuclear membrane is disassembled. Hence transcription cannot proceed until the cell undergoes mitotic division. This delay increases the probability of degradation of DNA.

Photonics4Life researcher Bavishna B. Praveen and his colleagues from the University of St Andrews found a trick to enhance the transfection efficiency: They use a synthetic peptide called Nupherin-neuron that helps the injected DNA from the cytoplasm into the nucleus. One of its ends consists of a nuclear localization signal which binds to the nucleus, while the other end, a cationic moiety, binds to the DNA. Using this helper, the researchers achieved a 3-fold enhancement in the transfection efficiency for adherent Chinese Hamster Ovary (CHO-K1) cells. Further, in the presence of this reagent, it was possible to reduce the required plasmid concentration by about 70% without compromising the transfection efficiency.

Perhaps even more important, the scientists for the first time demonstrated the transfection of non-adherent cells with very high efficiency, which is, in contrast to adherent cells, notoriously difficult. However, it is crucial for the development of high throughput microfluidic optical transfection devices, where a flow of cells in the microfluidic channel could be treated with femtosecond optical radiation.

J. Biophotonics 4(4), 229-235 (2011)

 

http://dx.doi.org/10.1002/jbio.201000105

Round Robin Experiment

Raman spectroscopy has already proved its effectiveness in many cases for medical diagnostics such as for cancer, cardiovascular diseases and infections. However, there are no standards in the different working groups, e.g. for sample preparation, implementation of the Raman experiments, spectra pre-treatment, data evaluation, etc.In a round robin experiment, the required groundwork will take place in order to define standardised Raman measurement methods, which will be fundamental for establishing Raman spectroscopy for clinical diagnostic procedures.

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