Establishment of a transient expression system for Dictyostelium discoideum.
We have established a rapid and sensitive transient expression system for Dictyostelium discoideum. We constructed a gene fusion containing the promoter from the Dictyostelium Actin 15 gene fused to the firefly luciferase gene. The enzymatic activity of this gene fusion, expressed at very high levels in stable transformants, was measured to determine optimum conditions for transient expression using electroporation to introduce the DNA into cells. With these conditions, we show that a luciferase gene fusion driven by a prestalk, cell-type specific promoter from the pst-cathepsin gene expresses luciferase at the appropriate developmental stage. In addition, we present results suggesting that the system will be useful for expressing genes in non-axenic cell lines. Finally, we observe that electroporation is more efficient for obtaining stable transformations than the standard calcium phosphate procedure using extrachromosomally replicating shuttle vectors but less efficient for vectors that integrate into the Dictyostelium chromosomes.
Howard
P K
PK
Department of Biology, University of California, San Diego, La Jolla 92093.
Ahern
K G
KG
Firtel
R A
RA
D000199:Actins / Q000235:genetics; D004023:Dictyostelium / Q000235:genetics*; D004560:Electricity; D005111:Extrachromosomal Inheritance; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D008156:Luciferases / Q000235:genetics; D011401:Promoter Regions, Genetic; D014162:Transfection
D000199:Actins; D008156:Luciferases