Open Access Highly Accessed Research article

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

Jannik Fonager1, Blandine MD Franke-Fayard1, John H Adams2, Jai Ramesar1, Onny Klop1, Shahid M Khan1, Chris J Janse1 and Andrew P Waters3*

Author Affiliations

1 Leiden Malaria Research Group, Department of Parasitology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden. The Netherlands

2 Department of Global Health, College of Public Health, University of South Florida, Tampa, Florida USA

3 Institute of, Infection, Immunity & Inflammation, School of Medical, Veterinary & Life Sciences, & Wellcome Centre for Molecular Parasitology, Glasgow Biomedical Research Centre, University of Glasgow, Scotland, UK

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BMC Genomics 2011, 12:155  doi:10.1186/1471-2164-12-155

Published: 20 March 2011

Additional files

Additional file 1:

Description of piggyBac insertions. Column 1: Origin of insertion described as: Parental population.clone.subclone (See main text for description). Column 2: Type of insertion in relation to CDS: 5' UTR, CDS, 3'UTR or more than 1 kb away from the nearest CDS (> 1 kb from CDS). Column 3: Insertion site distance to start ATG of CDS (for 5' UTR and CDS insertions) or from the end (stop codon) of the CDS (for 3' insertions). Column 4: Percentage of locus disrupted (for CDS insertions only) and calculated as the percentage of the locus (including introns) occurring after the insertion site. Column 5: Pb locus identifier as used by the Sanger Center (two first digits indicate chromosomal location on the 14 P. berghei chromosomes). Column 6: Insertion site sequence (TTAA) with the adjacent 20 5' and 3' nucleotides. Column 7: Gene description as provided by the Sanger Center.

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Additional file 2:

A characteristic of piggyBac inserts in 5'UTR and coding sequences (CDS and introns) of the P. berghei genome. Column 1: Type of insertion in relation to CDS: 5' UTR or CDS. Column 2: Pb locus identifier as used by the Sanger Center (two first digits indicate chromosomal location on the 14 P. berghei chromosomes). Column 3: Identifier for P. falciparum orthologs. Column 4: Gene description as provided by the Sanger Center. Column 5: Targeted disruption attempted (See column 8 for reference to study). Column 6: Exclusive expression in gametocytes/oocysts/sporozoites ([7,8]). Column 7: Other details regarding gene. Column 8: References to targeted disruption or expression of gene. Column 9: Origin of insertion described as: Parental population.clone.subclone (See main text for further description). Column 10: RMgmDB (RMgmDB; [42,22]) number assigned to disruption.

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Additional file 3:

Generation of parasites lacking expression of P41 (Δp41) or Metacaspase2 (Δmetacaspase2). (A) Schematic representation of the construct used for disruption of p41 and metacaspase2. Correct integration of the construct results in disruption of the genes as shown (replacement locus) and was analysed by Southern analysis of PFG-separated chromosomes and diagnostic PCR (see B and C). See (Additional file 5 Table S3) for primer details. Black boxes: target regions; grey box: tgdhfr-ts selectable marker cassette. (B) PCR analysis of correct disruption of p41, Northern analysis of transcription in wild type (wt) and mutant (318cl1) and Southern analysis of PFG-separated chromosomes. PCRs were performed with primers that amplify the 5' (INT1 and L313) region of the disrupted locus (5' int) or the intact ORF (INT1, INT2), c: Control PCR amplifying the p28 gene. Chromosomes were hybridized with the 3'-dhfr probe recognizing the p41-construct integrated in chromosome 10 and the endogenous dhfr-ts gene on chromosome 7. TM4 is a probe recognizing rRNA and used as a loading control. (C) PCR analysis of correct disruption of the metacaspase2 and Southern analysis of PFG-separated chromosomes in wild type (wt) and mutant (796cl1) (right-hand panel). PCRs were performed with primers that amplify the 5' (INT1 and 313) region of the disrupted locus (5' int) or the intact ORF (INT1, INT2), c: Control PCR amplifying the p28 gene. Chromosomes were hybridized with the 3'-dhfr probe recognizing the metacaspase2 construct integrated in chromosome 10, the endogenous dhfr-ts gene on chromosome 7 and the integrated GFP-construct in chromosome 3 of the parent line 507cl1.

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Additional file 4:

Location of intron within the 5'ITR of piggyBac. Green letters: gfp CDS. Black underlined letters: piggyBac 5' ITR. Black underlined bold letters: Intron.

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Additional file 5:

List of primers used in this study. Left column: primer number. Middle column: Primer sequence. Right column: Description of primer and orientation (F: Forward, R: Reverse).

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Additional file 6:

TAIL PCR conditions during Primary, Secondary and Tertiary rounds of PCR (adapted from [75]).

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