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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >High-efficiency transformation of Plasmodium falciparum by the lepidopteran transposable element piggyBac.
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High-efficiency transformation of Plasmodium falciparum by the lepidopteran transposable element piggyBac.

机译：鳞翅目转座因子piggyBac对恶性疟原虫的高效转化。

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摘要

Functional analysis of the Plasmodium falciparum genome is restricted because of the limited ability to genetically manipulate this important human pathogen. We have developed an efficient transposon-mediated insertional mutagenesis method much needed for high-throughput functional genomics of malaria parasites. A drug-selectable marker, human dihydrofolate reductase, added to the lepidopteran transposon piggyBac, transformed parasites by integration into the P. falciparum genome in the presence of a transposase-expressing helper plasmid. Multiple integrations occurred at the expected TTAA target sites throughout the genome of the parasite. We were able to transform P. falciparum with this piggyBac element at high frequencies, in the range of 10(-3), and obtain stable clones of insertional mutants in a few weeks instead of 6-12 months. Our results show that the piggyBac transposition system can be used as an efficient, random integration tool needed for large-scale, whole-genome mutagenesis of malaria parasites. The availability of such an adaptable genetic tool opens the way for much needed forward genetic approaches to study this lethal human parasite.
机译：由于遗传操纵这种重要的人类病原体的能力有限，因此恶性疟原虫基因组的功能分析受到限制。我们已经开发出一种高效的转座子介导的插入诱变方法，是疟原虫高通量功能基因组学所急需的。添加到鳞翅目转座子piggyBac中的药物选择标记人二氢叶酸还原酶通过在表达转座酶的辅助质粒存在下整合到恶性疟原虫基因组中而转化了寄生虫。在整个寄生虫基因组中的预期TTAA目标位点发生了多次整合。我们能够用这个piggyBac元件在10（-3）范围内高频转化恶性疟原虫，并在数周而不是6-12个月的时间内获得了稳定的插入突变体克隆。我们的结果表明，piggyBac转座系统可以用作大规模，全基因组诱变疟原虫所需的高效，随机整合工具。这种适应性强的遗传工具的可用性为研究这种致死性人类寄生虫的急需的正向遗传方法开辟了道路。

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