High-efficiency transformation of Plasmodium falciparum by the lepidopteran transposable element piggyBac
High-efficiency transformation of Plasmodium falciparum by the lepidopteran transposable element piggyBac
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DOI：
10.1073/pnas.0504679102
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发表时间：
2005-11-08
影响因子：
11.1
通讯作者：
Adams, JH
中科院分区：
文献类型：
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作者：
Balu, B;Shoue, DA;Adams, JH
由于对这种重要的人类病原体进行遗传操作的能力有限，因此对恶性疟原虫基因组的功能分析受到限制。我们开发了一种有效的转座子介导的插入诱变方法，这对于疟疾寄生虫的高通量功能基因组学非常需要。将药物选择标记人二氢叶酸还原酶添加到鳞翅目转座子piggyBac中，在表达转座酶的辅助质粒存在下，通过整合到恶性疟原虫基因组中来转化寄生虫。在整个寄生虫基因组的预期 TTAA 靶位点发生了多重整合。我们能够用该piggyBac元件以10(-3)范围内的高频率转化恶性疟原虫，并在几周而不是6-12个月内获得插入突变体的稳定克隆。我们的结果表明，piggyBac 转座系统可以用作疟原虫大规模全基因组诱变所需的高效、随机整合工具。这种适应性强的遗传工具的出现为研究这种致命的人类寄生虫急需的正向遗传方法开辟了道路。
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 A 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 A faliparum 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.