Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites
© Fonager et al; licensee BioMed Central Ltd. 2011
Received: 1 September 2010
Accepted: 20 March 2011
Published: 20 March 2011
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.
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.
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.
The sequencing of several Plasmodium genomes [1–4] has permitted large-scale microarray and proteomic studies of the different Plasmodium life cycle stages [3, 5–8]and comparative genomic analyses [9–12]. These studies have generated a wealth of information on the majority of the ~5.500 Plasmodium genes and have provided insight into the timing of expression during the lifecycle and into the putative function of many of the encoded proteins. However, almost one-half of the predicted genes still lack characterized orthologues in other systems and for most of these genes, the function remains unknown and the gene model unconfirmed. Reverse genetic approaches are often used to assign function to Plasmodium-specific genes. However, larger scale gene function analysis using reverse genetics in Plasmodium is hampered by the relative inefficiencies of genetic modification by targeted gene disruption or mutation [13, 14] and by the absence of other methods to modify gene expression such as RNAi gene silencing . Forward genetic approaches have not been widely applied in Plasmodium research because of the lack of adequate tools for whole genome analysis. Forward genetics is an experimental approach in which gene mapping and positional cloning are used to elucidate the molecular mechanisms underlying phenotypic differences between two individuals for a given trait. The advantage of this approach is that it involves an unbiased/random sampling of the genome, screening for a pre-determined phenotypic trait offering the possibility to identify multiple genes associated with a trait that directed reverse genetics is less likely to do. Often phenotypes are intentionally created by random mutagenesis using chemicals, radiation or insertional mutagenesis. Insertional mutagenesis has been at the core of functional genomics in many species. Transposable elements have been widely used to induce insertional mutagenesis in highly diverse biological systems and remain a mainstay for important model organisms. In a direct comparison of the four different transposable systems Sleeping-beauty, Tol2, Mos1 and piggyBac in four mammalian cell lines, piggyBac demonstrated significantly higher transposition activity in all lines . In Plasmodium, transposition has been reported using the Drosophila mariner transposable element but the transposition events occurred at a very low frequency independent of transposase . Recently, the piggyBac system has been successfully adapted for the human malaria parasite P. falciparum through the use of a two plasmid transfection approach: one transiently maintained plasmid containing the transposase and the other plasmid containing a positive selectable marker expression cassette flanked by the Inverted Terminal Repeat (ITR) sequences necessary for transposase mediated insertion. Parasites containing successful insertion events are drug-selected and this approach has enabled parasite efficient transformation by the piggyBac element [13, 18]. PiggyBac has now been successfully applied as a forward genetics tool using phenotypic screening of pools of P. falciparum mutants to identify genes that play a role in asexual blood stage development .
In this study we report the development of the piggyBac transposition system for the rodent malaria parasite P. berghei. The availability of relatively efficient reverse genetic technologies for P. berghei and the fact that these can be combined with analyses on parasites throughout their complete life cycle, both in vitro and in vivo, have made P. berghei the most frequently used model for gene function analysis [20–22]. The development of additional tools for analysis of gene function that would allow larger scale experiments would enhance gene function analysis in Plasmodium. The use of insertional mutagenesis as a tool for larger scale analysis of gene function is dependent on the efficiency of random insertion which in turn is dependent on: 1) the transfection efficiency of parasites for introducing the two plasmids, the piggyBac donor plasmid and the transient helper plasmid containing the transposase, in co-transfection experiments, 2) the activity in Plasmodium of a transposase which is evolved to function in insect cells and 3) the genome wide frequency, distribution and accessibility of the target TTAA site of integration. The P. berghei genome, like that of P. falciparum, is one of the most AT-rich of all eukaryotic genomes characterized (> 80% AT rising to >90% in non-coding and centromeric regions [1, 10]. Indeed, the P. falciparum genome is estimated to possess more than 300,000 TTAA sites with ~40% appearing in all characterized ESTs yielding an average of >20 integration sites per gene . Moreover, for P. berghei efficient transfection methods are available . Co-transfection of the transient helper plasmid and donor plasmid (as was also performed in P. falciparum), is commonly used to prevent unwanted piggyBac remobilization in the genome. However, it has been shown that constitutively active or regulated transposase expression can improve transposition efficiency up to 6 times . In this study we have investigated the frequency of random integration in parasites by expressing the transposase either transiently from introduced episomes or 'constitutively' from a transposase gene integrated into the genome. We observed efficient and random piggyBac-mediated insertion into the genome when parasites were transfected with piggyBac donor plasmids under both circumstances; either in combination with transposase-containing helper plasmids or in parasites containing the transposase gene stably integrated into the genome. We present evidence for piggyBac element remobilization in the latter parasites and therefore such parasites can be effective tools for generation of mutant parasite libraries containing random mutations. The availability of the technology for transposon-mediated random mutagenesis for P. berghei can be used to develop and apply large-scale forward genetic screens for analysing gene function.
Generation of piggyBac donor and helper plasmids and a transgenic P. berghei line containing transposase in its genome
The donor plasmid contains the 5'and 3' inverted terminal repeats of the piggyBac element (Figure 1A) which are the minimal cis elements necessary for piggyBac mobilization. Both inverted repeat sequences consist of a terminal 13 bp and internal 19 bp perfect inverted repeat that are separated by a 3 bp (5'ITR) or a 31 bp (3'ITR) spacer [26–30]. In the donor plasmid the two ITR sequences are located on both sides of a drug-selectable marker cassette and a gfp expression cassette that lacks a promoter region (Figure 1A). The target site for piggyBac insertion is TTAA and it moves by precise insertion and excision mechanisms [31, 32]. Transfection of the donor plasmid would therefore result in insertion of both the drug-selectable marker cassette and the gfp-expression. Insertion of the drug selectable marker, the human dhfr gene, allows for selection of parasites containing the inserts using pyrimethamine or WR99210.
Transfection of donor plasmids into parasites that either transiently or stably express transposase results in piggyBac-mediated insertion
Similarly, transfection of 3 different amounts (P3 = 15 μg; P4 = 10 μg; P5 = 5 μg) of the donor plasmid into TPSama1 parasites that stably express transposase, followed by selection with WR992210, resulted in selection of three resistant parasite populations (1182cl1m1-m3; parent population P3, P4 and P5). Southern analysis of FIGE-separated chromosomes of these parasites again showed integration of the donor plasmid (construct) in multiple chromosomes (Figure 2A, left panel). Based on the relative intensity of the hybridization signals it appears that a lower concentration of the donor plasmid results in higher insertion frequency. Analysing the Southern hybridization data of the FIGE-separated chromosomes from the 10 subpopulations of P5 (generated as described above) insertion of the constructs was observed in nearly all (groups) of chromosomes (Figure 2B, right panel).
PiggyBac insertion into all chromosomes
To identify the location of piggyBac insertions in the genome we initially used a standard method of inverse PCR using the forward primers 3202-3204 in combination with the reverse primers 3205-3207 as described for P. falciparum  to amplify re-ligated piggyBac sequences with P. berghei genomic flanking regions. However, this PCR method resulted in a very low yield of inserts when we used DNA extracted from parasites of the subpopulations. Compared to the number of insertions estimated based on the number positive chromosomes, we were able to retrieve less than 20% of the inserts from the different subpopulations (data not shown). We therefore decided to use an adapted method of TAIL-PCR. In this method a large pool of arbitrary degenerate primers designed for use in the AT rich genome of P. berghei (see Methods section) were used in combination with primers specific for both ITR's of the piggyBac element. By analyzing 9 subpopulations that had 16 visible inserts in the 14 P. berghei chromosomes, we were able to identify 11 inserts (~70%) from these chromosomes by TAIL-PCR. Therefore, for further identification of inserts we decided to exclusively use TAIL-PCR and no other methods such as inverse PCR or methods using restriction digestion and ligation . Using TAIL-PCR we identified insertions in parasites of parent population 2 and 5 and subpopulations of P1, P2 and P5. In total we identified 127 inserts at unique locations in the genome (Additional file 1 Table S1) and we have not identified any insert at the same location from two different parent populations, suggesting that there is no strong bias in preferred insertion sites between different experiments. However, for two genes: [GeneDB:PBANKA_060790] (in P2 and P5.b.3) and [GeneDB:PBANKA_082890] (in P2 and P5.i.3), insertions were identified both in 5'UTR/3'UTR and in CDS/3'UTR, respectively. In all 127 cases, that we identified by sequencing TAIL-PCR products, the insertion occurred via an expected canonical TTAA tetranucleotide (piggyBac) insertion site (Figure 2C, Additional file 1 Table S1).
Since the ability of piggyBac to randomly insert into the genome is an important feature of the piggyBac mutagenesis system in Plasmodium research, we investigated several aspects of the 127 insertions. First, we analysed the immediate 20 nucleotides adjacent to 5´ and 3´ flanking the TTAA site to evaluate if piggyBac insertion exhibited any additional preferences within the insertion flanking sequences. We compared these flanking regions with those of a set of 254 random chosen TTAA sequences from the genome (see Methods section). Overall, a slightly higher AT% was found in the piggyBac TTAA site flanking regions (81.7%) compared with the 254 random TTAA sites (80.4%), however this difference was not statistically significant (two tailed test, P = 0.13). A slight preference for a stretch of seven T's and three A's was observed at the 5´ and 3' of the TTAA piggyBac insertion sites (Figure 2C) when compared to the flanking regions of the randomly selected TTAA sites (Figure 2C). Both the 5'-stretch of seven T's and the 3'stretch A's at the piggyBac TTAA insertion site were significantly different from similar stretches of the random chosen TTAA sites (Chi Square: 5,5E-08 *** and 0,013* for the 5'and 3' stretches, respectively). This indicates that piggyBac may have a slight insertional bias with regard to the TTAA flanking sequences. We next analysed the chromosomal distribution of the 127 inserts. Three inserts (we term repetitive region 1-3) could not be mapped to a specific chromosome but were located on contigs containing genes that belong to known subtelomeric gene families, the bir or Pb-fam families of genes [10, 35]. The 124 remaining inserts were spread over all 14 chromosomes (Figure 2D, left panel) and a weak correlation (R2: 0.56) was observed between the number of inserts and the size of the chromosome. Chromosome 6 was exceptional as more than expected inserts (13) were identified in this chromosome compared to the number identified in the similar sized chromosomes 5, 7 and 8 in which 4-10 inserts were identified.
PiggyBac insertions into coding, untranslated and intergenic regions
The position of the 127 inserts in and around predicted genes show that piggyBac inserted both in coding sequence (CDS) and untranslated regions (UTR's) of genes (Figure 2D, right panel). For these regions a comparable proportion of piggyBac TTAA insertion sites and 254 randomly chosen TTAA sequences were observed: 24% (piggyBac integration) and 23% (random TTAA sites) in 5'UTR's; 45% and 54% in CDS (including introns); 23% and 16% in 3'UTRs; 8% and 7% in intergenic regions. For all regions the distribution of the piggyBac insertion sites was not significantly different from the random selected TTAA sites (Fisher's test: 5'UTR: p = 0.90, CDS (including introns): p = 0.15, 3'UTR: p = 0.12; intergenic regions: p = 0.84). These results indicate that piggyBac insertion in P. berghei occurs randomly and there is no preference for insertion either within the CDS or non-CDS of genes. We analysed whether piggyBac had a preference for insertion into transcribed/expressed genes, by analyzing published data on expression of genes with piggyBac insertions. For this analysis we used the new P. berghei gene models/systematic id's provided by the 'GeneDB 2010 release' and included only insertions that were located at a distance of more than 1 kb from the CDS (see Additional file 1 Table S1 for the included/excluded genes). Expression data were obtained from published proteomes from different P. berghei life cycle stages  and gametocytes . In addition, all available P. berghei EST databases present in the PlasmoDB database  were used (using the gene overlap function at default settings to retrieve ESTs matching genes assigned to different life cycle stages). Of the 124 analysed piggyBac inserts, 91 (73%) had proteome/EST based expression evidence compared with expression assigned to 3594 of the 4479 (80%) P. berghei gene models ('2010 Sanger P. berghei release'). Of the genes with piggyBac inserts, 55% (68 of 124) were transcribed/expressed in the asexual blood stages (ABS) compared to 54% of all P. berghei gene models (2420 of 4479) with evidence for transcription/expression in ABS (not significant, Fishers test: p = 0.07). Since genes that are inactive in ABS might be less accessible for piggyBac insertion, we compared the proportion of inserts in genes which are inactive in ABS but are expressed in other stages. There is transcription/proteome evidence that 26% (1174 of 4479) of all genes are expressed exclusively in stages other than the ABS. For genes with piggyBac inserts, 20% (25 of 124) of the genes are expressed in other life cycle stages but not in ABS (not significant, Fishers test: p = 0.15). In none of the comparisons mentioned above were significant differences found, indicating that piggyBac insertion is not linked to the expression pattern or activity of the genes.
A large proportion of the inserts were found inside CDS, CDS introns or in the 5'UTR regions (86 out of 127) of genes and such insertions most likely affect or completely disrupt the expression of these genes. The presence of an insert in the CDS/introns/5'UTR may therefore provide indirect evidence that the gene is not essential for ABS development. This is because Plasmodium is haploid during ABS development; therefore disruption of a gene essential for ABS would result in parasites that can not be selected after transfection as the deletion is lethal. We analysed 73 genes in more detail that contained inserts either in the CDS, CDS intron or in the 5'UTR with a maximum distance of 500 bp to the start codon of the CDS (Additional file 2 Table S2). Interestingly for at least 4 genes evidence already existed that expression of these genes is not essential in ABS as have been demonstrated by standard targeted deletion of these genes (See references in Additional file 2 Table S2). In addition, published data on expression in blood and mosquito stages [7, 8] showed that 8 genes (or their P. falciparum orthologs) are either gametocyte- or mosquito-stage specific and are not expressed in ABS, indicating that the lack of expression should have no effect on the survival of ABS. However, for many genes with piggyBac inserts evidence is available indicating expression of these genes in ABS (collated and available at PlasmoDB ) suggesting that these genes have a non-essential and/or redundant function during asexual blood stage development. We confirmed the non-essential role of two of such genes, p41 ([GeneDB :PBANKA_100260]; interrupted in its 5' UTR by piggyBac) and metacaspase2 ([GeneDB :PBANKA_130230]; interrupted in a CDS intron by piggyBac), through targeted disruption of these genes. P41 belongs to proteins encoded by the 6-cysteine family of genes and in P. berghei is transcribed in ABS . In P. falciparum P41 is intimately associated with GPI-anchored proteins that are located to the surface of merozoites (i.e. in lipid rafts). It has been reported that all P. falciparum GPI-anchored proteins (apart from MSP5) on the surface of merozoites are essential [38–40]. We targeted P. berghei p41 by disruption of the CDS through double cross-over recombination (Additional file 3 Figure S1) and confirmed that this protein was not essential in P. berghei ABS. The gene encoding P. falciparum metacaspase 2 [PF14_0363] is transcribed throughout ABS development [36, 41]. As with P41, we were able to disrupt the CDS of P. berghei metacaspase2 by targeted double cross-over recombination (Additional file 3 Figure S1), confirming the non-essential role of this protein in ABS. These data indicate that analysis of large scale piggyBac mutagenesis can provide evidence for the dispensability of certain, albeit ABS-expressed, genes for Plasmodium ABS. All 73 genes with piggyBac inserts in the CDS, CDS introns or in the 5'UTR region (within 500 bp from the start codon) have been deposited in the publically accessible database of genetically modified rodent malaria parasites (RMgmDB; [42, 22]) and this information on piggyBac insertion is linked to the information on individual genes in PlasmoDB  and GeneDB . For identification of the exact location of the insertion, the sequence of 20 bp up-and downstream of the TTAA sequence, is provided for all genes in RMgmDB  (See also Additional file 2 Table S2).
Evidence for re-mobilization of piggyBac inserts in parasites stably expressing transposase
The use of piggyBac to 'trap' Plasmodium promoters active in asexual blood stages
A total of 3 inserts were identified by TAIL-PCR survey in GFP-expressing parasites selected by FACS sorting. Two of these inserts occurred in a direction compatible with GFP expression (Figure 4D and See Additional file 1 Table S1). In addition, 2 inserts were identified from the P1e subpopulation derived by cloning of parent population P1 (See Table Additional file 1 Table S1 for inserts). The identification in a parasite population of one insert that is not compatible with GFP expression may indicate that this population contains parasites with gfp inserted into two different loci. FIGE-analysis of this population indeed shows evidence for integration into two loci, one in chromosome 6 and the other in one of the chromosomes of group 9-11 (Figure 4C). We determined transcription of gfp by Northern analysis in blood stages of the three FACS-sorted and P1e populations and in all parasites gfp transcripts were detected (Figure 4E). Transcription of gfp insert into the 5'UTR of gene [GeneDB :PBANKA_062360] (population F1/F3) was confirmed by RT-PCR (Figure 4F) demonstrating that our piggyBac approach had successfully trapped the promoter of this gene which belongs to the bir multi gene family. Interestingly, PCR analysis showed that the size of the RT-PCR amplified gfp-transcript in F1/F3 was smaller in comparison with the PCR-amplified genomic fragment (Figure 4F). Sequencing of the RT-PCR amplified product revealed that the smaller size was due to the unexpected removal of an intron in the piggyBac 5'ITR, not affecting the CDS of gfp (Additional file 4 Figure S2). This splicing event shows that the endogenous piggyBac 5'ITR can be recognized by the splicing machinery of P. berghei.
We have evaluated the efficiency and insertion characteristics of the piggyBac transposable element in the malaria parasite P. berghei. Insertional mutagenesis approaches have been widely used for genome characterization and transposon-mediated mutagenesis has become a powerful molecular genetic tool for eukaryotic transgenesis [46–50]. It has recently been shown that genomic insertional mutagenesis using piggyBac combined with a phenotypic screen for attenuated growth of the blood stages provided an effective tool for functional analysis of Plasmodium genes . Assuming that appropriate phenotypic screens can be devised, the availability of additional forward genetic technologies holds the great promise for large scale analysis of the function of the many 'hypothetical' Plasmodium proteins. Therefore, in this study we adapted the piggyBac system used for P. falciparum to P. berghei and developed it further. P. berghei, a rodent malaria parasite, is a frequently used model for the functional analysis of Plasmodium genes [10, 35, 51] and it allows for the analysis of Plasmodium gene function both in vitro and in vivo throughout the complete life cycle.
To obtain piggyBac insertions in the P. falciparum genome one million parasites were added to erythrocytes preloaded with the transposon donor and the transposase helper plasmid followed by selection of drug resistant parasite populations . In these 'parent populations' the number of insertions was low ranging between 1-14 as identified by inverse PCR or vectorette PCR reactions. Using these methods 177 unique piggyBac insertions have been identified in 81 independent transfections . To obtain piggyBac insertions in the P. berghei genome we used in this study the standard method of transfection of purified schizonts that result in high transfection efficiency of 10-2-10-3 when parasites are transfected with plasmids [21, 23]. PCR-based detection methods, such as TAIL PCR, have been shown to be highly efficient  and the method of choice in other organisms to identify piggyBac insertions [53–55]. We used an adapted TAIL PCR method here as traditional PCR was inadequate for these purposes in our hands. Using this TAIL PCR method we were able to detect 35 and 40 inserts, respectively, in the two parent populations (P5, P2) that were obtained by drug selection of 1-5 × 106 transfected parasites. These calculations indicate that in our studies 16 to 18 times more inserts could be identified per transfection experiment in P. berghei than currently reported for P. falciparum . It can be expected that a percentage of inserts generated by this approach will not have been identified since the Semi Arbitrary Degenerate (SAD) primers will exercise some specificity and the P. berghei genome is extremely AT-rich (< 80%). Furthermore inserts could have been missed since the ability to detect inserts with PCR based methods is highly dependent on the copy number of the insert . The development and use of additional SAD primer sets for Tail PCR in combination with sequencing strategies of increased efficiency/sensitivity might therefore lead to an increased number of identifiable insertions in the parent populations. In the subpopulations and cloned lines that were obtained from the parent populations by infection of mice with 1-5 parasites we estimated that we were able to identify approximately 65% of the inserts by Tail PCR. This is based on the comparison of the number of visible inserts detected in FIGE-separated chromosomes and the inserts identified by TAIL PCR. In total we have identified 127 inserts by TAIL PCR in parasites of only 2 transfection experiments (P2, P5), indicating that piggyBac integrates efficiently into the genome of P. berghei and that this model permits the generation of piggyBac insertion events significantly more efficiently than the human parasite, P. falciparum.
Analysis of the insert sites in the P. berghei genome showed that insertion occurred exclusively in the expected TTAA insertion site. Like in P. falciparum we found neither a (strong) bias for insertion into a particular chromosome nor a preference for insertion into transcribed/expressed genes indicating a random distribution of inserts. This is in contrast to piggyBac insertion into the genome of several other organisms, including mouse, zebrafish, Schistosoma, Drosophila and mammalian cell lines, where insertions predominantly occur into actively transcribed genes [57–60]. Interestingly we observed a slight insertional bias with regard to the sequence directly flanking the TTAA sequence with a slight preference of T's up- and A's downstream of the insertion site, respectively, which has also been observed in P. falciparum . On the other hand, we found no preference for insertion within or outside CDS, whereas in P. falciparum an increased number of insertions have been observed in the 5'UTR regions, which might also reflect preferential insertion into transcriptionally active genes or subtle differences in genome organization. The fact that piggyBac insertion into the P. berghei genome is for the most part a random process is important for the further development and application of this technology for larger scale forward genetic approaches.
As with P. falciparum we found that in the absence of transposase the piggyBac inserts remained stably integrated at the insertion sites even during prolonged periods of asexual multiplication (84 mitotic divisions in a 3 week period). It has been shown that piggyBac inserts can remobilize in genomes when transposase is present and several studies have estimated the rate of transposon remobilization [43, 61, 45, 60, 62–64]. When we introduced transposase stably into the genome under control of the ama-1 promoter remobilization of piggyBac inserts was detectable during blood stage asexual multiplication of cloned parasite lines. The observed rate of remobilization seems to be low as the majority of parasites before and after the period of asexual multiplication showed the same insert as judged by analysis of FIGE-separated chromosomes. In the three clones of parasite population 5.i we detected a total of 7-10 unique inserts in parasites that had multiplied for a period of 3 weeks (from the start of the cloning procedure). If we assume as above that we detected 65% of the inserts by TAIL PCR the rate of remobilization in these populations is around 15% per mitotic division (7-10 inserts per 84 mitotic divisions).
Remobilization might actually offer benefits by increasing the number of unique inserts in an experimental population towards the desired saturation levels of mutagenesis . For P. falciparum it has been calculated that ~15.000 mutations/inserts will represent about 50% saturation and obtaining such a level of saturation is seen as a difficult but realistic possibility for the P. falciparum genome . Remobilization could help to significantly reduce the number of transfections necessary to produce true saturation mutagenesis in a population. For instance, while a 50% coverage library would require ~380 individual transfections using the transient transposase expression strategy (~50 detectable transpositions per transfection) the same level could be obtained with far fewer transfections in parasites containing a stably expressed transposase. Since we observed a 7-10× increase in inserts during 3 mechanical blood passages as a result of remobilisation, a comparable 50% coverage could be obtained by as few as ~50 transfections that are passaged for a period of 3 weeks in mice. The integrated transposase in our experiments is controlled by the P. berghei ama-1 promoter, which is active only briefly in the schizont stage. Remobilisation will be especially beneficial if remobilisation can be controlled by regulating transposase activity. Encouragingly the use of inducible expression systems has been shown to greatly improve control of piggyBac insertion and remobilization rates in other organisms .
PiggyBac insertion into CDS or 5'UTRs of genes may provide indirect evidence that the gene is essential for blood stage development. Therefore the data on the location of inserts from large scale piggyBac mutagenesis experiments can provide additional evidence for the dispensability of Plasmodium genes for blood stage development, information that will be of use for example for validation of drug and vaccine targets. We confirmed the non-essential nature of two of the genes interrupted by piggyBac by standard targeted gene deletion. Of these pb41 is an orthologue of pf41 that encodes a GPI anchored protein found on the merozoite surface and as such as been proposed as a vaccine candidate. Strategies of vaccination targeting non-essential proteins have been attempted in the human infectious parasite P. knowlesi in the past and resulted in variant parasites that escaped the vaccination regime. In some cases the escaped parasites failed to express the target antigen . Therefore, knowledge of the essential nature of a protein proposed as a vaccine candidate is potentially significant. The use of the model P. berghei to determine the essential nature of conserved genes is relevant due to the relative ease of genetic manipulation in this system and in the cited example we have subsequently learned that PF41 is non-essential in P. falciparum (B. Crabb, personal communication).
We have therefore deposited all genes with piggyBac inserts in the CDS or in the 5'UTR region (500 bp from the start ATG) in the publically accessible database of genetically modified mutant parasites, RMgmDB  and this information on piggyBac insertion is linked to the information on individual genes in PlasmoDB  and GeneDB . In addition, we demonstrate that piggyBac insertion can be used to identify promoters that are active during blood stage development. FACS sorting of GFP expressing blood stage parasites appears to be an efficient method to collect parasites that have GFP inserted downstream of an active gene promoter region (i.e. 5'UTR) initiating GFP expression. One such identified insert was located in the promoter region of a member of the bir multigene family [GeneDB :PBANKA_062360]. FACS analysis of blood stage GFP expression of this FACS-sorted population, demonstrated a pattern of GFP expression that is highly comparable to expression of BIR proteins tagged with either GFP or mCherry, specifically showing highest levels of expression in maturing trophozoites and schizonts (results not shown). The most significant application of random mutagenesis is the ability to perform forward genetic screens to select mutants of a desired phenotype. Recently the feasibility of such an approach has been shown for P. falciparum by screening for mutants with attenuated growth of the blood stages. This relied upon parasite cloning and phenotype characterisation soon after piggyBac integration and screening method resulted in the identification of several parasite genes and pathways critical for intra-erythrocytic development . Such an approach is applicable to both P. falciparum and P. berghei offering the possibility to develop and apply forward genetic screens for additional and important phenotypes such as virulence, drug resistance, commitment to and successful completion of sexual development. In addition, phenotypic screens might be developed during mosquito transmission and pre-erythrocytic development. However, bottlenecks in parasite numbers during transmission in and out of the mosquito may reduce the efficiency of selecting the desired mutants from a large pool of piggyBac-mutants. For P. berghei efficient methods exist for production of gametocytes and ookinetes and production of mosquitoes containing large numbers of oocysts (> 500) and salivary gland sporozoites can relatively easily be scaled up. However, application of efficient phenotype screens during liver stage development will require the development of more efficient in vitro cultivation systems for the analysis of sporozoites into viable blood stage merozoites.
This study shows that piggyBac is an efficient and random transposon in P. berghei. In addition, piggyBac is able to remobilize from genomic P. berghei insertion sites, which could, with further development facilitate the generation of saturated mutagenesis lines for use in forward genetic screens.
Generation of the piggyBac donor and the helper plasmids for transient expression of transposase
The pXL-BACII-GFP-HDHFR piggyBac transposon donor plasmid (pL1302) was created by cloning a gfp-expression cassette without promoter region and a hdhfr selectable marker cassette into the minimal piggyBac vector pXL-BACII . The gfp-expression cassette, containing gfpm3 and the 3'pbdhfr/ts was excised from plasmid pL0024 by BamHI/EcoRI digestion while digesting the pXL-BACII vector with BglII/EcoRI resulting in the intermediate plasmid pXL-BACII- gfpm3-3'pbdhfr flanked by XhoI/EcoRI restriction sites. The hdfr selectable marker cassette (5' pbeef1aa-hdhfr-3'pbdhfr) was excised from plasmid pL0009 with EcoRI digestion and subcloned into the intermediate pXL-BACII- gfpm3-3'pbdhfr plasmid, generating the final piggyBac donor plasmid, pXL-BACII-gfp-hdhfr. (pL1302). The pHTH transient helper plasmid (pL1301) was constructed by first excising the piggyBac transposase coding sequence from the pHTH plasmid used for P. falciparum ; kindly provided by Bharath Balu) with BamHI and cloned into the intermediate plasmid pL0011 under the control of 5'-pbeef1aa and 3'pbdhfr, resulting in plasmid pL1303. The 5' pbeef1aa-transposase-3'pbdhfr cassette was excised from pL1303 with BamHI digestion and subcloned into the plasmid pL0004, generating pHTHpb (pL1301) for transient transposase expression. The plasmids pL0004, pL0009, pL0011 and pL0024 can be obtained from MR4 .
Construction of a plasmid to generate a transgenic P. berghei line stably expressing transposase
To stably integrate the piggyBac transposase gene into the P. berghei genome plasmid pL1307 was generated. The transposase coding sequence was excised from the pHTH plasmid (see above) with BamHI and cloned into the expression plasmid pL0010 (MR4; ), which contains the toxoplasma gondii dhfr-ts encoding selectable marker cassette and the dssu-rrna target sequence for single cross-over integration into the non-essential c/d-rrna locus locus. The transposase was cloned with BamHI into the 5'pbama1 - 3'pbdhfr expression cassette of pL0010.
Transfection and selection of parasites
Transfection, selection and cloning of mutant parasite lines were performed as described . For experiments for transient transfection of parasites with the transposase-containing helper plasmid, circular donor plasmid pL1302 and helper plasmid pL1301 were mixed in a 1:1 (exp. 1056) or 1:2 (exp. 1056) ratios using 5 μg donor plasmid per transfection prior to transfection of the parasites (P. berghei ANKA, cl15cy1). Selection of resistant parasite populations after transfection was performed by treatment of the infected mice with pyrimethamine. As a control parasites were transfected with 5 μg donor plasmid (exp 1057). For generation of the transgenic line containing transposase stably integrated into the genome, parasites (P. berghei ANKA, cl15cy1) were transfected with plasmid pL1307 after linearization with Apa-1. Selection and cloning of transgenic parasites were performed as described . One clone, 1042cl1 (TPSama1) was used for further experiments. After transfection of TPSama1 with donor plasmid pL1302 a resistant parasite population (1182) was selected by treatment with WR9221 . The genotype of transfected parasites was analysed by standard PCR analysis and Southern blot analysis of digested genomic DNA or of FIGE separated chromosomes .
Analysis of transcription: Northern blot and RT-PCR analysis
RNA extraction and Northern blot analysis was performed according to standard methods [68, 69]. RNA of mixed blood stages was hybridized to a gfp-probe that was PCR amplified from plasmid pL1302 using the primer set 3552-3553 (Additional file 5 Table S3) that is specific for the gfp coding sequence (CDS). As a loading control, the ethidium bromide stained gel was used. RNA used for RT-PCR analysis was first digested with DNaseI for 1 hour, followed by phenol/chloroform extraction. Reverse transcription was performed using SuperScriptIII™ (Invitrogen) according to the manufacturer's instructions after splitting the RNA into two samples, one to which reverse transcriptase was added (+RT) and one without (-RT). RT-PCR was performed using primer 4571 targeting a P. berghei genomic sequence located 117 bp upstream of the confirmed piggyBac insertion site in 5' UTR of the bir gene [GeneDB :PBANKA_062360]. Primer 4571 was used in combination with primer 3209, located in the gfp CDS of the piggyBac insert (442 bp from the terminal end of the 5'ITR; see Additional file 5 Table S3 for primer sequences). The expected PCR product size on gDNA using the primer pair 4571/3209 is 559 bp. As a control for reverse transcription and DNaseI digestion, the gene [GeneDB :PBANKA_133840] was amplified from both cDNA and genomic DNA with the primer set 3902/3903 located on either side of two confirmed introns (expected sizes are 530 bp on gDNA and 232 bp on cDNA). Thirty-five PCR amplification cycles were performed with Amplitaq DNA polymerase (Roche) using 4 mM MgCl2 and an annealing temperature of 50°C and 1 minute extension time at 72°C.
FACS sorting and analysis of GFP expressing parasites
For FACS sorting of GFP-positive parasites, we collected 10 μl of infected tail blood (with a parasitemia of 3%) in complete culture medium from mice infected with parasites from exp. 1056 (see above). GFP-positive cells were sorted on the FACSAria Cell Sorter (Becton Dickinson). Selection of blood cells on forward/side light scatter and filter settings for detection of GFP-fluorescence was performed as described . GFP-positive cells were sorted using three different gates as shown in the Results Section (Figure 4A) and sorted cells (50-300 cells per sorting experiment) were collected in ~300 μl complete culture medium at room temperature. This cell-suspension was injected intravenously (tail vein) into a single mouse. 7-8 days after injection, parasites were collected from the mice and were analysed for GFP expression. GFP-fluorescence intensity of live blood stages was examined after staining with Hoechst-33258  using a Leica-fluorescence MDR microscope and pictures recorded using a DC500 digital camera.
Thermal Asymmetric Interlaced (TAIL) PCR and identification of insertion sites
To identify piggyBac inserts, two sets of each three primers specific for the piggyBac 5' and 3'ITR regions (Additional file 5 Table S3) were used successively in combination with 8 Semi-Arbitrary Degenerate (SAD) primers (Additional file 5 Table S3) with 140 to 192 fold degeneracy. Each SAD primer contains a Sau3Ai site (4 with GATC and 4 with the reverse complement CTAG at the 3' end) since this sequence is widely distributed in the P. berghei genome (approximately 2.3 × 104 occurrences identified by text searches against all contigs present in PlasmoDB . The SAD primers were designed to reflect the variability surrounding the GATC sites in P. berghei from a multiple alignment of around 1100 genomic P. berghei sequences obtained from PlasmoDB . TAIL-PCR was performed as described [52, 72] with some modifications of the protocol (See Additional file 6 Table S4 for the different conditions used for TAIL-PCR). Briefly, eight primary TAIL-PCR reactions were performed, each with one of the SAD primers (3649-3656) and in combination with either the piggyBac 5'ITR primer 3202 or the 3'ITR primer 3726. After the initial PCR reaction, products were diluted 1:2000 with distilled water and used in the secondary reaction, containing the piggyBac primers 3203 (5'ITR) or 3727 (3'ITR) together with the same SAD primer as used in the primary reaction. The secondary PCR products were diluted 1:500 and used in the tertiary reaction mixture. The tertiary PCR reaction was performed with piggyBac primers 3204 (5'ITR) or 3728 (3'ITR) again with the same SAD primers as before. The tertiary amplification fragments were visualized through electrophoresis in 1% agarose gels, where the average fragment size was usually between 300 and 500 bp. Immediately after the tertiary reaction, the reaction mixture was purified using a PCR-purification kit (Roche Applied Science, Germany) according to manufacturers instructions. Subsequently the reaction mixture (containing several TAIL PCR products) was ligated into the TOPO TA vector (Invitrogen) and transformation and blue/white screening was performed according to manufacturer's instructions. DNA from 15 to 20 of positive colonies were analysed by sequencing. The insertion site in the genome of the piggyBac cassette was determined by BLASTN (vs. PlasmoDB  and GeneDB  databases) analysis of the sequences outside of the piggyBac cassette followed by precise identification of the insertion site using BioEdit alignment and mapping (using Artemis) of each TAIL-PCR sequence to the closest gene. If an insertion occurred more than 1000 bp from the CDS of a gene, it was not considered to be located in its (potential) 5' or 3'UTRs. For comparison of characteristics of the piggyBac insertion sites, 254 randomly TTAA sites and flanking regions were extracted from P. berghei genomic sequences (obtained from the Sanger Centre ). Visualization of the TTAA insertion site and flanking site conservation was performed with Weblogo 3.0 .
Expression profiles of genes with piggyBac insertions
Using the Boolean operators, implemented in the PlasmoDB site , sets of genes with proteome/EST evidence were generated using the databases implemented on the PlasmoDB site  including published P. berghei gametocyte and proteomes from all developmental stages [3, 7]. All datasets were converted to the updated gene models available (released by Sanger Centre as of 15 April 2010) and a comparison was made between all expressed/transcribed genes and genes with piggyBac insertions for each set.
Targeted disruption of p41 and metacaspase2
Two replacement DNA constructs were made for targeted disruption of p41 [GeneDB :PBANKA_100260] and metacaspase2 [GeneDB :PBANKA_130230] by double cross-over homologous recombination. Details of primers used to generate double-cross over replacement constructs (i.e. to introduce 'targeting regions' in the replacement construct pL0001; (See MR4  and also Additional file 3 Figure S1 and Additional file 5 Table S3). The p41 gene has been disrupted in the ANKA reference line cl15cy1  and metacaspase2 has been disrupted in the GFP-expressing reference line of the ANKA strain (line 507cl1, identifier RMgm-7 in ). Transfection, selection and cloning of mutant parasites was performed as described . Correct integration of the constructs was analysed by diagnostic PCR and Southern analysis of FIGE-separated chromosomes and transcription by standard Northern analysis of mRNA collected from mixed blood stage parasites . Primers for amplification of the probes used for Northern analysis are shown (See Additional file 3 Figure S1 and Additional file 5 Table S3). As a loading control Northern blots were hybridized with primer L644R that hybridizes to the blood stage large subunit ribosomal RNA .
This work was supported by The Netherlands Organization for Scientific Research (ZonMw TOP grant number 9120_6135 to JF); and The National Institutes of Health (R01 AI33656, R21 AI070888 to JHA); and The European Commission (FP6 BioMalPar Network of Excellence; FP7 EviMalar Network of Excellence); and The Wellcome Trust.
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