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. 2011 Mar 20:12:155.
doi: 10.1186/1471-2164-12-155.

Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Affiliations

Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Jannik Fonager et al. BMC Genomics. .

Abstract

Background: The genome of a number of species of malaria parasites (Plasmodium spp.) has been sequenced in the hope of identifying new drug and vaccine targets. However, almost one-half of predicted Plasmodium genes are annotated as hypothetical and are difficult to analyse in bulk due to the inefficiency of current reverse genetic methodologies for Plasmodium. Recently, it has been shown that the transposase piggyBac integrates at random into the genome of the human malaria parasite P. falciparum offering the possibility to develop forward genetic screens to analyse Plasmodium gene function. This study reports the development and application of the piggyBac transposition system for the rodent malaria parasite P. berghei and the evaluation of its potential as a tool in forward genetic studies. P. berghei is the most frequently used malaria parasite model in gene function analysis since phenotype screens throughout the complete Plasmodium life cycle are possible both in vitro and in vivo.

Results: We demonstrate that piggyBac based gene inactivation and promoter-trapping is both easier and more efficient in P. berghei than in the human malaria parasite, P. falciparum. Random piggyBac-mediated insertion into genes was achieved after parasites were transfected with the piggyBac 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 piggyBac element in parasites that stably express transposase was demonstrated.

Conclusion: These data demonstrate that piggyBac behaved as an efficient and random transposon in P. berghei. Remobilization of piggyBac element shows that with further development the piggyBac system can be an effective tool to generate random genome-wide mutation parasite libraries, for use in large-scale phenotype screens in vitro and in vivo.

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Figures

Figure 1
Figure 1
The piggyBac insertion (donor) construct and helper constructs for expression of transposase. A. The helper plasmid pHTH (pL1301, left) contains the transposase gene under control of the constitutive eef1aa promoter and the dhfr/ts 3'UTR for transient transposase expression and the donor plasmid (pL1302, right) contains the gfp-expression cassette without a promoter and the hdhfr selectable marker cassette. Both cassettes are flanked by the piggyBac inverted terminal repeats (ITR's). B. Schematic representation of the construct pL1307 for stable integration of the transposase gene (under the control of the ama1 promoter) into the P. berghei genome in the non-essential small subunit ribosomal rna gene (ssu-rrna) of the c/d-rrna unit. SM: the tgdhfr/ts selectable marker cassette. Primers used for diagnostic PCRs are indicated by arrows with the expected fragment size (see C). lsu: large subunit, ets: external transcribed spacer region. C. Diagnostic PCR and FIGE analysis of separated chromosomes of mutant TPSama1 confirming correct integration of construct pL1307 into the rrna gene locus. See B for the location of the primers; 537/538 control primers for the p28 locus; (Additional file 3 Figure S1).
Figure 2
Figure 2
Location of PiggyBac inserts into the genome of P. berghei. A. PiggyBac insertions as shown by FIGE analysis of separated chromosomes hybridized with the pbdhfr/ts probe. This probe recognizes the inserts and the endogenous dhfr/ts gene on chromosome 7 (arrow). Left panel: Inserts in two parent parasite populations (1, 2) after transient transfection of the helper (h) and donor (d) plasmid (h/d ratio: P1 = 1:1; P2 = 1:2; c = control parasites transfected with only donor plasmid). Right panel: insertions in 15 (a to o) parasite subpopulations of P1. B. PiggyBac insertions as shown by FIGE analysis of separated chromosomes hybridized with the pbdhfr/ts probe, which recognizes the endogenous dhfr/ts gene (chromosome 7) and the transposase construct pL1307 in chromosome 5 (arrows). Left panel: Insertions in the three parent parasite populations after transient transfection of the donor (d) plasmid into parasites of mutant TPSama1 that contains transposase integrated into chromosome 5 (amount of d: P3 = 15 μg; P4 = 10 μg; P5 = 5 μg). Right panel: insertions in 10 parasite subpopulations of P5 (a to l). C. Upper panel: WebLogo representation of the sequence of 127 piggyBac insertion sites, showing the TTAA insertion site and 20 bp up- and downstream of the piggyBac 5'ITR and 3'ITR, respectively. Lower panel: WebLogo representation of 20 bp up- and down-stream sequence of 254 randomly chosen TTAA sites in the P. berghei genome. D. Left panel: Chromosomal distribution of the 254 randomly chosen TTAA sites and 124 piggyBac TTAA insertion sites. Right panel: location of the piggyBac inserts (black bars) and 254 random TTAA sites (white bars) in CDS (+introns), within 1 kb 5' or 1 kb 3' to the CDS (designated as 5' UTR and 3 'UTR) or in the intergenic regions (> 1 kb from CDS).
Figure 3
Figure 3
PiggyBac insertions in cloned parasites with transient or stable expression of transposase. A. PiggyBac insertions as shown by FIGE analysis of separated chromosomes hybridized with the pbdhfr/ts or hdhfr probe. The pbdhfr/ts probe recognizes the inserted constructs, the endogenous dhfr/ts gene on chromosome 7 and the transposase construct pL1307 integrated into chromosome 5 (arrows). Without stable expression of transposase a single insert is detected in parasite clones both before and after prolonged periods of multiplication (i.e. after mechanical (am) passage; see B. In parasite clones that stably express transposase multiple inserts are detected in different chromosomes, both before and after mechanical passage (see B). B. Number of piggyBac inserts as determined by TAIL PCR in cloned parasite lines before and after prolonged periods of multiplication (i.e. after mechanical passage). ND: No data.
Figure 4
Figure 4
Identification of piggyBac-trapped promoters. A. FACS sorting of GFP-expressing parasites from a population with piggyBac inserts. Dot plots show GFP fluorescence and forward light scatter of (infected) erythrocytes of a control population without inserts (left plot) and of parent population P2 (see Figure 2A) with piggyBac inserts (right plot). Three populations (F1-F3) of GFP-expressing parasites were collected by sorting from gates F1 - F3. B. GFP-expression in blood stages of FACS-sorted populations F1-F3 and P1.5e (see Figure 2A). Gates: g1: infected erythrocytes (Hoechst positive) that are GFP-negative; g2: infected erythrocytes (Hoechst positive) that are GFP-positive. Percentages (mean + st. dev.) of GFP-positive infected cells: Control) 0.1%; F1) 41.6% + 3; F2) 4.0% + 0.7; F3) 28.1% + 2.9; P1e) 1. 6% + 0.2). C. PiggyBac insertions in GFP-expressing parasites of the F1-F3 populations shown by Southern analysis of chromosomes hybridized with pbdhfr/ts and gfp probes. D. Schematic representations of two piggyBac insertion sites identified by TAIL-PCR in the GFP-expressing parasites of the F1-F3 populations. Insertion location is compatible with GFP expression (black arrows; see also Additional file 1: Table S1). Grey arrow: location of integration primer (4571). E. Northern analysis of gfp-expression in blood stages of the Control and F1-F3 populations and subpopulation P1.5e. Loading control: ethidium bromide stained RNA. F. Confirmation of gfp transcription from the bir gene promoter in population F1 by RT-PCR (upper panel; see also Methods section; gDNA and cDNA (+RT or -RT enzyme) were obtained from F1 or Control parasites). Lanes: 1) Marker; 2) gDNA-F1; 3) cDNA-F1; 4) gDNA-Control; 5) cDNA-Control; 6) No DNA. Control of cDNA quality (lower panel) was performed by PCR across the two introns of the gene PBANKA_133840 (GeneDB [34]]). Lanes: 1) Marker 2) gDNA-Control; 3) cDNA-Control; 4) No cDNA-Control; 5) cDNA -F1; 6) No cDNA-F1; 7) No DNA.

References

    1. Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW. et al.Genome sequence of the human malaria parasite Plasmodium falciparum. Nature. 2002;419:498–511. doi: 10.1038/nature01097. - DOI - PMC - PubMed
    1. Pain A, Bohme U, Berry AE, Mungall K, Finn RD, Jackson AP. et al.The genome of the simian and human malaria parasite Plasmodium knowlesi. Nature. 2008;455:799–803. doi: 10.1038/nature07306. - DOI - PMC - PubMed
    1. Hall N, Karras M, Raine JD, Carlton JM, Kooij TW, Berriman M. et al.A comprehensive survey of the Plasmodium life cycle by genomic, transcriptomic, and proteomic analyses. Science. 2005;307:82–86. doi: 10.1126/science.1103717. - DOI - PubMed
    1. Carlton JM, Angiuoli SV, Suh BB, Kooij TW, Pertea M, Silva JC. et al.Genome sequence and comparative analysis of the model rodent malaria parasite Plasmodium yoelii yoelii. Nature. 2002;419:512–519. doi: 10.1038/nature01099. - DOI - PubMed
    1. Bozdech Z, Llinas M, Pulliam BL, Wong ED, Zhu J, DeRisi JL. The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol. 2003;1:E5. doi: 10.1371/journal.pbio.0000005. - DOI - PMC - PubMed

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