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).