Optimum conditions for electric pulse-mediated gene transfer to mammalian cells in suspension.

Abstract

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

Molecular Identification of a Human Carcinoma‐associated Glycoprotein Antigen Recognized by Mouse Monoclonal Antibody FU‐MK‐1

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Improvement of DNA transfection with cationic liposomes

“…Electric pulses or electroporation The physical variables in the electric pulse-mediated transfer of DNA into cells (40,46) are the duration of the electric pulse T (5-20 msec) and the intensity of the field. Duration depends on the resistance of the medium (Q) and the potency (uF) of the capacitor (T == Q .…”

Cloning and expression of phospholipase A2 from guinea pig gastric mucosa, its induction by carbachol and secretion in vivo

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