Gaudet Pascale, Pilcher Karen E, Fey Petra, Chisholm Rex L
dictyBase, Center for Genetic Medicine, Northwestern University, 676 North Saint Clair Street Suite 1260, Chicago, Illinois 60611, USA.
Nat Protoc. 2007;2(6):1317-24. doi: 10.1038/nprot.2007.179.
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.
DNA介导的转化是研究基因功能最广泛使用的技术之一。真核生物盘基网柄菌适合进行多种需要将外源DNA插入细胞的基因操作。在这里，我们描述两种常用的转化盘基网柄菌细胞的方法：磷酸钙沉淀法，可产生高拷贝数的转化体；以及电穿孔法，这是一种将单个整合事件导入基因组DNA的有效技术。通过同源重组进行基因敲除需要单整合。我们还讨论了不同的选择标记如何影响转化体中的载体拷贝数，并解释了为什么杀稻瘟菌素已成为制作基因敲除的首选选择标记。这两种方法都可以在不到2小时的实际操作时间内完成；然而，磷酸钙沉淀法包含几次孵育，其中一次至少4小时，因此转化所需的总时间约为8小时。
