Centromere plasmid: a new genetic tool for the study of Plasmodium falciparum.
Centromere plasmid: a new genetic tool for the study of Plasmodium falciparum.
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Centromere质粒：用于研究恶性疟原虫的新遗传工具。
DOI：
10.1371/journal.pone.0033326
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Yuda M
中科院分区：
文献类型：
--
作者：
Iwanaga S;Kato T;Kaneko I;Yuda M
将转基因引入恶性疟原虫（一种高毒力的人类疟疾寄生虫），要么是通过单次交叉重组，要么是通过使用寄生质粒。然而，由于转染效率低和重组频率低，这些技术仍然不足。为了改进恶性疟原虫的遗传操作，我们开发了着丝粒质粒作为一种新的遗传工具。首先，我们试图将所有预测的恶性疟原虫着丝粒克隆到大肠杆菌细胞中，但由于这些序列的高A/T含量而失败。为了克服这一困难，我们确定了疟原虫着丝粒的共同序列特征，并设计了一个保留这些特征的小着丝粒。用小着丝粒序列构建的着丝粒质粒pFCEN在子代寄生虫中分离的效率约为99%，即使在没有药物选择的情况下，该质粒也能在恶性疟原虫中保持稳定。这一结果表明pFCEN中的小着丝粒序列可以作为恶性疟原虫的真正着丝粒发挥作用。此外，与不含着丝粒序列的对照质粒相比，使用pFCEN的转基因寄生虫的生成速度更快。此外，与对照质粒相比，pFCEN不形成串联体，因此在多次细胞分裂中保持单一拷贝。pFCEN质粒的这些独特性质将解决目前恶性疟原虫遗传操作的技术限制，因此，该质粒将成为研究该寄生虫的标准遗传工具。
The introduction of transgenes into Plasmodium falciparum, a highly virulent human malaria parasite, has been conducted either by single crossover recombination or by using episomal plasmids. However, these techniques remain insufficient because of the low transfection efficiency and the low frequency of recombination. To improve the genetic manipulation of P. falciparum, we developed the centromere plasmid as a new genetic tool. First, we attempted to clone all of the predicted centromeres from P. falciparum into E. coli cells but failed because of the high A/T contents of these sequences. To overcome this difficulty, we identified the common sequence features of the centromere of Plasmodium spp. and designed a small centromere that retained those features. The centromere plasmid constructed with the small centromere sequence, pFCEN, segregated into daughter parasites with approximately 99% efficiency, resulting in the stable maintenance of this plasmid in P. falciparum even in the absence of drug selection. This result demonstrated that the small centromere sequence harboured in pFCEN could function as an actual centromere in P. falciparum. In addition, transgenic parasites were more rapidly generated when using pFCEN than when using the control plasmid, which did not contain the centromere sequence. Furthermore, in contrast to the control plasmid, pFCEN did not form concatemers and, thus, was maintained as a single copy over multiple cell divisions. These unique properties of the pFCEN plasmid will solve the current technical limitations of the genetic manipulation of P. falciparum, and thus, this plasmid will become a standard genetic tool for the study of this parasite.
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影响因子：
6.7
作者：
Campbell TL;De Silva EK;Olszewski KL;Elemento O;Llinás M
通讯作者：
Llinás M
影响因子：
14.9
作者：
Deitsch, KW;Driskill, CL;Wellems, TE
通讯作者：
Wellems, TE
影响因子：
14.9
作者：
O'Donnell, RA;Preiser, PR;Crabb, BS
通讯作者：
Crabb, BS
影响因子：
16
作者：
Xiao, Hua;Mizuguchi, Gaku;Wu, Carl
通讯作者：
Wu, Carl
DOI：
10.1073/pnas.92.4.973
发表时间：
1995-02-14
影响因子：
11.1
作者：
WU, YM;SIFRI, CD;WELLEMS, TE
通讯作者：
WELLEMS, TE