Has the time come for us to complement our malaria parasites?

Goldberg, D. E., Janse, C.J., Cowman, A. F. and Waters, A.P. (2010) Has the time come for us to complement our malaria parasites? Trends in Parasitology, 27(1), pp. 1-2. (doi: 10.1016/j.pt.2010.06.017)

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Publisher's URL: http://dx.doi.org/10.1016/j.pt.2010.06.017


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], [5] and [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 brought to bear on a hypothesis, the more solid the conclusions are. Therefore, we propose the following: 1. Genetic complementation of Plasmodium knockout mutants is highly desirable and should be attempted whenever possible. Sexual crosses can be considered a form of genetic complementation for Plasmodium berghei [15]. 2. If complementation is technically infeasible or impractical, at least a second independent mutant must be analyzed. No research should be reported where the conclusions rest on the analysis of a single cloned mutant. 3. Restoration of the wild-type phenotype as a result of reversion of single crossover disruptants can be taken as suggestive but not definitive evidence for a knockout phenotype, as it is possible for an unanticipated phenotype-causing mutation to revert during culture. 4. Unambiguous evidence of knockout and complementation should be provided. Southern analysis (PFG-separated chromosomes and/or restricted genomic DNA) should be considered to be the gold standard. PCR evidence is suggestive but can be misleading. Evidence for loss of expression (northern or ideally western blots) increases confidence in the results. We believe that adherence to these standards will enrich the malaria field and put it on a par with those of other genetically manipulable organisms.

Item Type:Articles
Glasgow Author(s) Enlighten ID:Waters, Professor Andy
Authors: Goldberg, D. E., Janse, C.J., Cowman, A. F., and Waters, A.P.
College/School:College of Medical Veterinary and Life Sciences > School of Infection & Immunity
Journal Name:Trends in Parasitology

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