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).
Fonager et al. BMC Genomics 2011 12:155 doi:10.1186/1471-2164-12-155