Has the time come for us to complement our malaria parasites?
Has the time come for us to complement our malaria parasites?
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我们是时候补充我们的疟疾寄生虫了吗？
DOI：
10.1016/j.pt.2010.06.017
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发表时间：
2011
影响因子：
9.6
通讯作者：
Waters,AndrewP
中科院分区：
文献类型：
--
作者：
Goldberg,DanielE;Janse,ChrisJ;Cowman,AlanF;Waters,AndrewP
疟疾寄生虫的基因改造正变得越来越容易，可用的操作范围正在缓慢增加。根据物种的不同，我们现在可以进行单交换和双交换同源重组[1]，在复制、转录或蛋白质稳定性水平上调节表达[2]，阶段特异性重组[3]和转座子插入[4-6]。文献中报道的基因敲除的数量现在有数百个[7]，其中许多具有有趣的表型，可以告知基因功能。2001年报道了敲除克隆的第一次互补[8]，但从那时起，只进行了少数遗传互补。这种忽略的危险在于，被操纵的寄生虫在选择过程中可能会发生意想不到的突变[9]，从而导致观察到的表型。事实上，突变体的表型可能是由于基因座的破坏导致的非预期的位置效应。这一点在其他系统中早已为人所知;在许多酵母和细菌系统中，互补是必需的，现在也可以将这一原理应用于顶复门寄生虫疟原虫和弓形虫。对于恶性疟原虫，已建立的选择性标记仅限于二氢叶酸还原酶[10]和杀稻瘟菌素S-脱氨酶[11]。也许主要的障碍是从这些富含AT的基因组中克隆全长编码区的困难。获得正确的表达水平和时机也可能是有问题的[12]。对于啮齿类疟疾寄生虫，互补可能需要使用许多额外的动物，因为在缺乏多个真正独立的选择标记的情况下，载体再循环是唯一现实的途径[13]。尽管如此，我们认为，当它可以完成时，应该进行互补，因为它增加了表型是预期操作的功能的信心。关于突变寄生虫基因型和表型报告标准化的一些问题也在本期的另一篇文章中讨论，该文章描述了所有转基因啮齿动物疟原虫的数据库[14]。就像所有科学一样，越多的证据
Genetic modification of malaria parasites is becoming more facile and the range of available manipulations is slowly increasing. Depending on the species, we can now do single and double crossover homologous recombination [1], regulated expression modulated at the replication, transcription or protein stability levels [2], stage-specific recombination [3] and transposon insertion [4–6]. The number of gene knockouts reported in the literature is now in the hundreds [7], many with interesting phenotypes that inform on gene function. The first complementation of a knockout clone was reported in 2001 [8], but since then only a handful of genetic complementations have been performed. The peril in this omission is that the manipulated parasites could have unintended mutations during selection [9] that contribute to the phenotype observed. Indeed, the phenotype of a mutant might be due to an unintended positional effect resulting from disruption of a locus. This has been known for a long time in other systems; complementation is de rigeur in many yeast and bacterial systems and it is now also possible to apply this principle to the Apicomplexan parasites Plasmodium and Toxoplasma.Complementation is still not easy in malaria parasites. For Plasmodium falciparum, established selectable markers are limited to dihydrofolate reductase [10] and blasticidin S-deaminase [11]. Perhaps the major hurdle is the difficulty in cloning full-length coding regions from these AT-rich genomes. Getting the right level and timing of expression can also be problematic [12]. For rodent malaria parasites, complementation can require use of many extra animals, as vector recycling is the only realistic route in the absence of multiple truly independent selectable markers [13]. Nevertheless, we would argue that when it can be done, complementation should be done, as it increases confidence that the phenotype is a function of the intended manipulation. Some concerns relating to standardization in reporting genotypes and phenotypes of mutant parasites are also discussed in a separate article in this issue, which describes a database of all genetically modified rodent malaria parasites [14]. As in all science, the more evidence