Development of the <it>piggyBac </it>transposable system for <it>Plasmodium berghei </it>and its application for random mutagenesis in malaria parasites
<p>Abstract</p> <p>Background</p> <p>The genome of a number of species of malaria parasites (<it>Plasmodium </it>spp.) has been sequenced in the hope of identifying new drug and vaccine targets. However, almost one-half of predicted <it>Plasmodium <...
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BMC
2011-03-01
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| Series: | BMC Genomics |
| Online Access: | http://www.biomedcentral.com/1471-2164/12/155 |
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| author | Janse Chris J Khan Shahid M Klop Onny Ramesar Jai Adams John H Franke-Fayard Blandine MD Fonager Jannik Waters Andrew P |
| author_facet | Janse Chris J Khan Shahid M Klop Onny Ramesar Jai Adams John H Franke-Fayard Blandine MD Fonager Jannik Waters Andrew P |
| author_sort | Janse Chris J |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>The genome of a number of species of malaria parasites (<it>Plasmodium </it>spp.) has been sequenced in the hope of identifying new drug and vaccine targets. However, almost one-half of predicted <it>Plasmodium </it>genes are annotated as hypothetical and are difficult to analyse in bulk due to the inefficiency of current reverse genetic methodologies for <it>Plasmodium</it>. Recently, it has been shown that the transposase <it>piggyBac </it>integrates at random into the genome of the human malaria parasite <it>P. falciparum </it>offering the possibility to develop forward genetic screens to analyse <it>Plasmodium </it>gene function. This study reports the development and application of the <it>piggyBac </it>transposition system for the rodent malaria parasite <it>P. berghei </it>and the evaluation of its potential as a tool in forward genetic studies. <it>P. berghei </it>is the most frequently used malaria parasite model in gene function analysis since phenotype screens throughout the complete <it>Plasmodium </it>life cycle are possible both in vitro and in vivo.</p> <p>Results</p> <p>We demonstrate that <it>piggyBac </it>based gene inactivation and promoter-trapping is both easier and more efficient in <it>P. berghei </it>than in the human malaria parasite, <it>P. falciparum</it>. Random <it>piggyBac</it>-mediated insertion into genes was achieved after parasites were transfected with the <it>piggyBac </it>donor plasmid either when transposase was expressed either from a helper plasmid or a stably integrated gene in the genome. Characterization of more than 120 insertion sites demonstrated that more than 70 most likely affect gene expression classifying their protein products as non-essential for asexual blood stage development. The non-essential nature of two of these genes was confirmed by targeted gene deletion one of which encodes P41, an ortholog of a human malaria vaccine candidate. Importantly for future development of whole genome phenotypic screens the remobilization of the <it>piggyBac </it>element in parasites that stably express transposase was demonstrated.</p> <p>Conclusion</p> <p>These data demonstrate that <it>piggyBac </it>behaved as an efficient and random transposon in <it>P. berghei</it>. Remobilization <it>of piggyBac </it>element shows that with further development the <it>piggyBac </it>system can be an effective tool to generate random genome-wide mutation parasite libraries, for use in large-scale phenotype screens <it>in vitro </it>and <it>in vivo</it>.</p> |
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| spelling | doaj.art-bf9a045205ee4f88be9e2342d31ec9192022-12-21T18:48:55ZengBMCBMC Genomics1471-21642011-03-0112115510.1186/1471-2164-12-155Development of the <it>piggyBac </it>transposable system for <it>Plasmodium berghei </it>and its application for random mutagenesis in malaria parasitesJanse Chris JKhan Shahid MKlop OnnyRamesar JaiAdams John HFranke-Fayard Blandine MDFonager JannikWaters Andrew P<p>Abstract</p> <p>Background</p> <p>The genome of a number of species of malaria parasites (<it>Plasmodium </it>spp.) has been sequenced in the hope of identifying new drug and vaccine targets. However, almost one-half of predicted <it>Plasmodium </it>genes are annotated as hypothetical and are difficult to analyse in bulk due to the inefficiency of current reverse genetic methodologies for <it>Plasmodium</it>. Recently, it has been shown that the transposase <it>piggyBac </it>integrates at random into the genome of the human malaria parasite <it>P. falciparum </it>offering the possibility to develop forward genetic screens to analyse <it>Plasmodium </it>gene function. This study reports the development and application of the <it>piggyBac </it>transposition system for the rodent malaria parasite <it>P. berghei </it>and the evaluation of its potential as a tool in forward genetic studies. <it>P. berghei </it>is the most frequently used malaria parasite model in gene function analysis since phenotype screens throughout the complete <it>Plasmodium </it>life cycle are possible both in vitro and in vivo.</p> <p>Results</p> <p>We demonstrate that <it>piggyBac </it>based gene inactivation and promoter-trapping is both easier and more efficient in <it>P. berghei </it>than in the human malaria parasite, <it>P. falciparum</it>. Random <it>piggyBac</it>-mediated insertion into genes was achieved after parasites were transfected with the <it>piggyBac </it>donor plasmid either when transposase was expressed either from a helper plasmid or a stably integrated gene in the genome. Characterization of more than 120 insertion sites demonstrated that more than 70 most likely affect gene expression classifying their protein products as non-essential for asexual blood stage development. The non-essential nature of two of these genes was confirmed by targeted gene deletion one of which encodes P41, an ortholog of a human malaria vaccine candidate. Importantly for future development of whole genome phenotypic screens the remobilization of the <it>piggyBac </it>element in parasites that stably express transposase was demonstrated.</p> <p>Conclusion</p> <p>These data demonstrate that <it>piggyBac </it>behaved as an efficient and random transposon in <it>P. berghei</it>. Remobilization <it>of piggyBac </it>element shows that with further development the <it>piggyBac </it>system can be an effective tool to generate random genome-wide mutation parasite libraries, for use in large-scale phenotype screens <it>in vitro </it>and <it>in vivo</it>.</p>http://www.biomedcentral.com/1471-2164/12/155 |
| spellingShingle | Janse Chris J Khan Shahid M Klop Onny Ramesar Jai Adams John H Franke-Fayard Blandine MD Fonager Jannik Waters Andrew P Development of the <it>piggyBac </it>transposable system for <it>Plasmodium berghei </it>and its application for random mutagenesis in malaria parasites BMC Genomics |
| title | Development of the <it>piggyBac </it>transposable system for <it>Plasmodium berghei </it>and its application for random mutagenesis in malaria parasites |
| title_full | Development of the <it>piggyBac </it>transposable system for <it>Plasmodium berghei </it>and its application for random mutagenesis in malaria parasites |
| title_fullStr | Development of the <it>piggyBac </it>transposable system for <it>Plasmodium berghei </it>and its application for random mutagenesis in malaria parasites |
| title_full_unstemmed | Development of the <it>piggyBac </it>transposable system for <it>Plasmodium berghei </it>and its application for random mutagenesis in malaria parasites |
| title_short | Development of the <it>piggyBac </it>transposable system for <it>Plasmodium berghei </it>and its application for random mutagenesis in malaria parasites |
| title_sort | development of the it piggybac it transposable system for it plasmodium berghei it and its application for random mutagenesis in malaria parasites |
| url | http://www.biomedcentral.com/1471-2164/12/155 |
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