piggyBac is an effective tool for functional analysis of the Plasmodium falciparumgenome

Abstract

BackgroundMuch of the Plasmodium falciparum genome encodes hypothetical proteins with limited homology to other organisms. A lack of robust tools for genetic manipulation of the parasite limits functional analysis of these hypothetical proteins and other aspects of the Plasmodium genome. Transposon mutagenesis has been used widely to identify gene functions in many organisms and would be extremely valuable for functional analysis of the Plasmodium genome.ResultsIn this study, we investigated the lepidopteran transposon, piggyBac, as a molecular genetic tool for functional characterization of the Plasmodium falciparum genome. Through multiple transfections, we generated 177 unique P. falciparum mutant clones with mostly single piggyBac insertions in their genomes. Analysis of piggyBac insertion sites revealed random insertions into the P. falciparum genome, in regards to gene expression in parasite life cycle stages and functional categories. We further explored the possibility of forward genetic studies in P. falciparum with a phenotypic screen for attenuated growth, which identified several parasite genes and pathways critical for intra-erythrocytic development.ConclusionOur results clearly demonstrate that piggyBac is a novel, indispensable tool for forward functional genomics in P. falciparum that will help better understand parasite biology and accelerate drug and vaccine development.

piggyBacTransposon

“…One of the best examples of mutagenesis using piggyBac is in vivo mutagenesis in mice for cancer gene discovery. Traditionally, murine leukemia virus and murine mammary tumor virus were used (80,107,135) for oncogene discovery in hematopoietic cells (71,72) and mammary tissues (73), respectively. However, such experiments were not possible in a large fraction of solid tumors due to the limited accessibility of these retroviruses.…”

Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum

“…With the lack of RNAi technology in this parasite, forward genetic approaches suitable for large-scale functional analysis are needed to validate possible drug targets and to gain a better understanding of P. falciparum pathophysiology (1). Recent efforts aimed to develop such tools include the use of Piggy-Bac, peptide-conjugated morpholino oligomers (PPMO), zinc-finger nucleases, glmS ribozyme, CRISPR-Cas9-mediated genome editing, peptide nucleic acids, and the Tet-R aptamer system (2)(3)(4)(5)(6)(7)(8). Each of these methods requires further development and optimization to be used in a large-scale format to assess the function of P. falciparum genes.…”

CCR4-Associated Factor 1 Coordinates the Expression of Plasmodium falciparum Egress and Invasion Proteins

“…Members of the CCR4-Not complex are well conserved in all Plasmodium species, suggestive of their significance in parasite gene regulation as well (10). In this study, we use a Plasmodium falciparum caf1 disruptant (⌬CAF1) mutant, created during the course of a large-scale transposon mutagenesis project of P. falciparum (3), to evaluate the function of the CCR4-Not complex in parasite gene regulation and its significance in intraerythrocytic development.…”