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Published February 2005 | public
Journal Article

Estimating the number of plasmids taken up by a eukaryotic cell during transfection and evidence that antisense RNA abolishes gene expression in Physarum polycephalum

Abstract

We have estimated the statistical distribution of the number of plasmids taken up by individual Jurkat lymphoma cells during electroporation in the presence of two plasmids, one encoding for yellow (EYFP) the other for cyan (ECFP) fluorescent protein. The plasmid concentration at which most of the cells take up only one plasmid or several molecules was determined by statistical analysis. We found that cells behaved slightly heterogeneous in plasmid uptake and describe how the homogeneity of a cell population can be quantified by Poisson statistics in order to identify experimental conditions that yield homogeneously transfection-competent cell populations. The experimental procedure worked out with Jurkat cells was applied to assay the effectiveness of antisense RNA in knocking down gene expression in Physarum polycephalum. Double transfection of flagellates with vectors encoding EYFP and antisense-EYFP revealed for the first time that gene expression can be suppressed by co-expression of antisense RNA in Physarum. Quantitative analysis revealed that one copy of antisense expressing gene per EYFP gene was sufficient to completely suppress formation of the EYFP protein in Physarum.

Additional Information

© 2004 Federation of European Microbiological Societies. Received 12 July 2004; received in revised form 15 October 2004; accepted 18 November 2004. First published online 8 December 2004. Edited by D.P. Wakelin We thank Mrs. Monika Messerschmidt and Mrs. Hannelore Jonas for excellent technical assistance. We are indebted to Drs. Stefan Kircher and Tim Kunkel for providing microscopy facilities.

Additional details

Created:
August 19, 2023
Modified:
October 25, 2023