Howard P K, Ahern K G, Firtel R A
Department of Biology, University of California, San Diego, La Jolla 92093.
Nucleic Acids Res. 1988 Mar 25;16(6):2613-23. doi: 10.1093/nar/16.6.2613.
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.
我们已经为盘基网柄菌建立了一个快速且灵敏的瞬时表达系统。我们构建了一个基因融合体，其中包含来自盘基网柄菌肌动蛋白15基因的启动子与萤火虫荧光素酶基因融合。该基因融合体在稳定转化体中以非常高的水平表达，通过电穿孔将DNA导入细胞来测定其酶活性，以确定瞬时表达的最佳条件。在这些条件下，我们表明由pst - 组织蛋白酶基因的前柄细胞类型特异性启动子驱动的荧光素酶基因融合体在适当的发育阶段表达荧光素酶。此外，我们给出的结果表明该系统将有助于在非无菌细胞系中表达基因。最后，我们观察到，使用染色体外复制穿梭载体时，电穿孔获得稳定转化的效率比标准磷酸钙程序更高，但对于整合到盘基网柄菌染色体中的载体，电穿孔的效率较低。
