Coplanar film electrodes facilitate bovine nuclear transfer cloning
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Automated lab on chip systems offer increased throughput and reproducibility, but the implementation of microelectrodes presently relies on miniaturisation of parallel plate electrodes that are time consuming and costly to fabricate. Electric field modelling of open electrofusion chambers suggested that widely spaced (≥2 mm) coplanar film electrodes should result in similar cell fusion rates as parallel plate electrodes provided the cell positioning was roughly midway between the electrodes. This hypothesis was investigated by electrofusion trials of bovine oocyte-donor cell couplets used in nuclear transfer (NT) cloning. Comparative experiments with reference parallel plate electrodes were conducted as controls. Coplanar fusion rates ≥ 90% were demonstrated for embryonic blastomeres, follicular cells and fetal and adult fibroblasts as NT donor cells. For embryonic and adult cell types, there was no significant difference in fusion rate between coplanar and parallel plate electrodes. For both electrode geometries, fusion efficiency with adult fibroblasts was highest at a calculated field strength of 2.33 kV/cm. The coplanar electrodes required a voltage π/2 times greater than parallel plate electrodes to achieve equivalent field strength when the couplets are placed midway between the electrodes.
KeywordsCloning Bovine Nuclear transfer Electrofusion
We would like to thank T. Delaney, J. Oliver, J. Oswald and P. Turner for excellent technical assistance in carrying out the fusion experiments; S. Finlay and M. Steyn-Ross, University of Waikato, for loan of equipment and technical advice; and H. Devereux, University of Canterbury, for advice on micro fabrication.
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