Estimating the number of plasmids taken up by a eukaryotic cell during transfection and evidence that antisense RNA abolishes gene expression in Physarum polycephalum.
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.
Materna
Stefan C
SC
Institut für BiologieIII, Albert-Ludwigs-Universität, Schänzlestrasse 1, 79104 Freiburg im Breisgau, Germany.
Marwan
Wolfgang
W
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D018274:Electroporation; D005057:Eukaryotic Cells / Q000737:chemistry; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells / Q000737:chemistry*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D016839:Physarum polycephalum / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D016012:Poisson Distribution; D016372:RNA, Antisense / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014170:Transformation, Genetic
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D016372:RNA, Antisense; D012333:RNA, Messenger; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins