Quantification of DNA uptake by Dictyostelium discoideum amoebae and the stability of the DNA during growth and development.
We have developed a simple and accurate method to determine the amount of intact plasmid DNA taken up and retained by Dictyostelium discoideum amoebae during various transformation protocols. We have used this method to compare the efficiency of three different methods for introducing foreign DNA into D. discoideum amoebae. Both a calcium phosphate and a spheroplast fusion procedure gave good uptake, but no intracellular plasmid DNA was detectable after calcium chloride treatment. The exogenous DNA was rapidly lost after transformation but was 20-fold more stable during starvation rather than growth conditions, suggesting a possible approach to improving transformation efficiency. No transient expression of neomycin phosphotransferase activity of any of the heterologous animal or plant promoters used could be detected using a sensitive gel assay procedure.
D004271:DNA, Fungal / Q000378:metabolism*; D004023:Dictyostelium / Q000235:genetics / Q000254:growth & development*; D010957:Plasmids; D014170:Transformation, Genetic
D004271:DNA, Fungal