ESTABLISHMENT OF A TRANSIENT EXPRESSION SYSTEM FOR DICTYOSTELIUM-DISCOIDEUM
ESTABLISHMENT OF A TRANSIENT EXPRESSION SYSTEM FOR DICTYOSTELIUM-DISCOIDEUM
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DOI：
10.1093/nar/16.6.2613
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发表时间：
1988-03-25
影响因子：
14.9
通讯作者：
FIRTEL, RA
中科院分区：
文献类型：
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作者：
HOWARD, PK;AHERN, KG;FIRTEL, RA
我们建立了一种快速、灵敏的盘基网柄菌瞬时表达系统。我们构建了一个融合了鞭毛菌肌动蛋白15基因启动子和萤火虫荧光素酶基因的融合基因。在稳定的转化子中高水平表达的该基因融合的酶活性被测量，以确定利用电穿孔将DNA导入细胞进行瞬时表达的最佳条件。在这些条件下，我们证明了由PST-组织蛋白基因的前柄、细胞型特异性启动子驱动的荧光素酶基因融合在适当的发育阶段表达荧光素酶。此外，我们提出的结果表明，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，与使用染色体外复制穿梭载体的标准钙磷酸盐程序相比，电穿孔技术在获得稳定转化方面更有效，但对于整合到Dictyostelius染色体中的载体来说效率较低。
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.