falciparumclones. The plasmids pLBacII-HDH-GFP and pLBacII-HDH-eGFP can trap promoters in the genome if inserted in the right orientation downstream to an endogenous promoter as shown previously [31]. These plasmids can also be

modified for stable transgene expression with or without GFP tag. Parasites transformed with pLBacII-HDGH, with hDHFR-GFP fusion as find more selectable marker, display high levels of fluorescence and are amenable to sorting by Fluorescence activated cell sorter (FACS). Transformation with the plasmid pLBacII-HDH-KanOri inserts the kanamycin resistance gene and a pUC origin of replication into the parasite genome that allows for plasmid rescue from the genome for easy identification of insertion sites. The genome-wide integration ofpiggyBacinto genes in all functional categories, expressed in all parasite life cycle stages, validates its application

in whole-genome mutagenesis ofP. falciparum. Almost all mutantP. falciparumclones generated had singlepiggyBacinsertions in their genomes, which will aid in easy correlation of mutant phenotypes to their respective genotypes. The increased number of insertions obtained in 5′ UTRs of genes indicates either active changes in chromatin structure allow easy access forpiggyBacto the genomic DNA or the affinity of the transposase for chromatin associated factors unique Bcl-2 inhibitor to these regions. Alternatively, this skewed distribution could simply be the inability to recover LY2606368 price mutants with insertions in coding Tacrolimus (FK506) sequences of essential genes, whereas insertions in 5′ UTRs of essential genes may not completely abolish gene expression and hence may not be lethal. From whole-genome mutagenesis perspectives, insertions in 5′ UTRs may have a varied effect on neighbouring gene expression. Insertions in 5′ UTRs

could either increase gene expression, possibly due to better recruitment of transcription machinery, or decrease gene expression by blocking transcription. A meaningful approach would therefore be to subject all 5′ UTR mutants to phenotypic analyses as either increased or decreased gene expression can significantly alter intracellular activities. Such a scenario might be particularly beneficial in identifying essential genes that cannot be knocked out in the parasite. Nevertheless, 22% of the insertions were obtained in coding sequences generating 39 gene knockouts, which almost equal the number of unique gene knockouts generated inP. falciparumthus far until a recent large-scale study achieving 53 gene knockouts [32], using conventional methods [10]. Such high propensity to create gene disruptions and the ability to rapidly generate stable lines of mutant clones, warrants the use ofpiggyBacin large-scale mutagenesis studies not only to identify gene functions, but also to discriminate the essential and dispensable regions of the parasite genome that will further confine the search for potent drug targets.