A rapid and efficient method to generate multiple gene disruptions in Dictyostelium discoideum using a single selectable marker and the Cre-loxP system -: art. no. e143
A rapid and efficient method to generate multiple gene disruptions in Dictyostelium discoideum using a single selectable marker and the Cre-loxP system -: art. no. e143
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DOI：
10.1093/nar/gnh136
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发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
Kimmel, AR
中科院分区：
文献类型：
--
作者：
Faix, J;Kreppel, L;Kimmel, AR
盘基网柄菌已被证明是一个非常强大的系统，用于研究细胞和发育功能的许多方面。网柄网柄菌相对较小(类似于34Mb)的染色体基因组和高效率的靶向基因干扰使研究人员能够表征许多特定的基因功能。然而，网柄菌中可选标记的数量受到限制，菌株之间进行有效遗传杂交的能力也是有限的。因此，很难在单个细胞内创造多个突变来研究基因之间的上位关系或不同途径之间的潜在冗余。我们现在描述了一个强大的系统，通过循环使用Cre-loxP系统回收单一的可选择标记--杀青菌素S抗性，来产生网柄苔藓的多个基因突变。我们通过产生携带四个独立基因中断的单个细胞来证实该系统的有效性。此外，细胞对需要选择杀菌素的额外靶向或随机突变以及使用其他可选择标记进行突变或标记蛋白的功能表达研究的转化保持敏感。
Dictyostelium discoideum has proven an exceptionally powerful system for studying numerous aspects of cellular and developmental functions. The relatively small (similar to34 Mb) chromosomal genome of Dictyostelium and high efficiency of targeted gene disruption have enabled researchers to characterize many specific gene functions. However, the number of selectable markers in Dictyostelium is restricted, as is the ability to perform effective genetic crosses between strains. Thus, it has been difficult to create multiple mutations within an individual cell to study epistatic relationships among genes or potential redundancies between various pathways. We now describe a robust system for the production of multiple gene mutations in Dictyostelium by recycling a single selectable marker, Blasticidin S resistance, using the Cre-loxP system. We confirm the effectiveness of the system by generating a single cell carrying four separate gene disruptions. Furthermore, the cells remain sensitive to transformation for additional targeted or random mutagenesis requiring Blasticidin selection and for functional expression studies of mutated or tagged proteins using other selectable markers.