Transformation of Dictyostelium discoideum with plasmid DNA
Transformation of Dictyostelium discoideum with plasmid DNA
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DOI：
10.1038/nprot.2007.179
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Chisholm, Rex L.
中科院分区：
文献类型：
--
作者：
Gaudet, Pascale;Pilcher, Karen E.;Chisholm, Rex L.
DNA转化是基因功能研究中应用最广泛的技术之一。真核生物盘基网眼菌能够经受许多需要将外源DNA插入细胞内的遗传操作。在这里，我们描述了两种常用的转化Dictyostelials细胞的方法：磷酸钙沉淀法，产生高拷贝数的转化子；以及电穿孔，一种产生单一整合事件到基因组DNA的有效技术。同源重组的基因破坏需要单一的整合。我们还讨论了不同的选择标记如何影响转化子中的载体拷贝数，并解释了为什么blastiidin成为进行基因敲除的首选选择标记。这两个过程都可以在不到2小时的实际操作时间内完成；然而，磷酸钙沉淀法包含几个孵化阶段，其中一个至少需要4个小时，因此转化所需的总时间大约为8个小时。
DNA-mediated transformation is one of the most widely used techniques to study gene function. The eukaryote Dictyostelium discoideum is amenable to numerous genetic manipulations that require insertion of foreign DNA into cells. Here we describe two commonly used methods to transform Dictyostelium cells: calcium phosphate precipitation, resulting in high copy number transformants; and electroporation, an effective technique for producing single integration events into genomic DNA. Single integrations are required for gene disruption by homologous recombination. We also discuss how different selection markers affect vector copy number in transformants and explain why blasticidin has become the preferred selectable marker for making gene knockouts. Both procedures can be accomplished in less than 2 h of hands-on time; however, the calcium phosphate precipitation method contains several incubations, including one of at least 4 h, so the total time required for the transformation is approximately 8 h.