A pulse-generating machine which delivers exponentially decaying pulses over broad range of pulse lengths was used to determine the optimum pulse conditions for gene transfer to FM3A cells. In the transformation of tk-cells with pTK1, a single pulse of 100-2000 ƒÊs gave a high transformation frequency at 1.5-6 kV/cm and room temperature, the highest transformation frequency obtained being 3 •~ 10-3. As the suspension buffer for cells exposed to the pulse, Saline G was better than PBS(-) for obtaining a large number of transformants because it ensured high cell viability. DNA transfer is a fundamental techniques used for the molecular cloning of genes and to study the expression and integration of foreign genes in mammalian cells. Recently devised electric pulse-mediated gene transfer, electroporation, is effective for both adhering (5, 9, 11) and floating cells (7, 10). We obtained a transformation frequency of 2 •~ 10-4 with floating FM3A mouse cells suspended in phosphate-buffered saline by using a high voltage semi-rectangular pulse (1). We here describe a newly constructed generator which delivers exponentially decaying pulses over broad range of decaying time constants (pulse lengths) and its use for DNA-mediated gene transfer. We also report the relation between cell survival and transformation in comparison to electric field strength, pulse length and composition of suspension buffers. From the results of the pulse experiments we determined the optimum conditions for gene transfer. MATERIALS AND METHODS Cell strain and culture conditions. A temperature-sensitive variant of FM3A, tsFT20tk-(4), was grown in RPMI 1640 medium supplemented with 10 % calf serum (Gibco) in an incubator (33•Ž and 5 % CO2). Preparation of plasmid DNA. The plasmid pTK1 (3) DNAs were prepared by the Toray Research Center, Kanagawa, Japan as described elsewhere (1) and were digested with restriction endonuclease Hind III to obtain a linear form. Apparatus for electroporation. A high voltage generator that can supply exponentially decaying electric pulses (initial voltage : V 0 =0-4 kV) was constructed. Decaying time
Since bacteria grow in high temperature and high humidity, bacterial food poisoning frequently occurs from the rainy season to summer. In Japan, the number of food poisoning cases is high from June to October. Maintaining a hygienic environment in the kitchen is very important for preventing food poisoning. In particular, cutting boards on which various foods are places may cause secondary pollution. Therefore, to avoid food poisoning, this study compared the ATP value of the cutting board before and after the hygiene education using the ATP wiping test and investigated the educational effect. Before hygiene education, the inspector conducted an ATP wiping test on the cutting boards for vegetables and meat that washed before and after cooking and notified the cooks of the values. The inspector conducted hygiene education while showing the cook how to clean the cutting board. The cutting board washed with detergent and sponge, rinsed with running water for 30 seconds or more, then this process was repeated twice.
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