A Genetic Tool for Future Cures
The tiny malaria parasite, one of humanity's oldest foes, is finally yielding its secrets to cutting-edge genetic technology.
Explore the ResearchMalaria remains one of humanity's most persistent health challenges, claiming hundreds of thousands of lives annually. Understanding the complex biology of Plasmodium parasites—the microorganisms that cause malaria—is crucial for developing new treatments and vaccines. Yet, nearly half of Plasmodium genes have no known function, presenting a massive scientific blind spot.
This article explores how scientists are using a sophisticated genetic tool, the piggyBac transposon system, combined with next-generation sequencing, to crack the parasite's genetic code in the rodent malaria model Plasmodium berghei—a breakthrough that could accelerate the discovery of new drug targets.
Annual malaria deaths worldwide
Plasmodium genes with unknown function
AT-rich Plasmodium genome
For decades, malaria researchers faced a frustrating limitation: the inability to efficiently determine which parasite genes are essential for survival and infection. Traditional reverse genetics approaches, where scientists disrupt specific genes to study their function, have proven extremely challenging and inefficient in Plasmodium parasites due to low transfection efficiency.
Start with a gene and look for its function by disrupting it
Start with random mutations and identify those causing interesting phenotypes
"The piggyBac transposon system provides a powerful forward genetics tool to study gene function in Plasmodium parasites via random insertion mutagenesis and phenotypic screening," note researchers who pioneered this approach 1 .
Until recently, however, identifying where these genetic insertions occurred in the parasite's genome remained tedious and inefficient, creating a major bottleneck in malaria research.
The piggyBac transposon, originally discovered in the cabbage looper moth, is a mobile genetic element that can efficiently cut and paste itself into genomes. This natural genetic wanderer has been developed as a versatile tool for manipulating genomes across diverse species, from insects to mammals 3 .
What makes piggyBac particularly useful for malaria research is its targeting preference—it inserts specifically into TTAA sites in DNA. The Plasmodium genome is exceptionally AT-rich (over 80%), meaning it contains hundreds of thousands of these TTAA sites, with approximately 20 potential integration sites per gene 3 .
This abundance of targets enables widespread mutagenesis across nearly the entire genome.
When piggyBac inserts into a gene, it can disrupt that gene's function, allowing scientists to determine what role it might play in the parasite's life cycle. If parasites with an insertion in a particular gene cannot complete their development in mosquito hosts, for instance, that gene becomes a promising candidate for new transmission-blocking drugs or vaccines.
Now used across diverse species
Insertion site sequence
Previous methods for identifying piggyBac insertion sites relied on various PCR-based techniques that were laborious and low-yield. A breakthrough came in 2013 when researchers demonstrated that next-generation sequencing could revolutionize this process 1 .
Scientists co-transfected P. berghei parasites with two types of plasmids: a donor plasmid containing the piggyBac transposon flanked by inverted terminal repeats, and a helper plasmid providing the transposase enzyme that facilitates the "cut and paste" movement into the genome 1 2 .
After transfection, parasites underwent drug selection to eliminate those without successful piggyBac insertions. The researchers then extracted genomic DNA from the resistant parasite population for analysis 2 .
The critical innovation came in how they identified insertion sites. While they initially used the traditional inverse PCR method, they primarily leveraged the power of Illumina HiSeq sequencing to rapidly sequence the entire genome of the mutated parasites 1 .
The contrast between traditional methods and the new sequencing approach was dramatic:
| Method | Number of Insertions Identified | Key Limitations |
|---|---|---|
| Inverse PCR | 10 | Tedious, low efficiency, multiple steps |
| Next-generation sequencing | 47 | Requires specialized equipment and bioinformatics expertise |
The power of next-generation sequencing didn't stop at identifying piggyBac insertions. As a bonus, researchers also discovered 1,850 single nucleotide polymorphisms (SNPs) between their laboratory P. berghei ANKA strain and the reference genome sequence 1 —valuable information for understanding genetic variation in research parasites.
| Target site | TTAA tetranucleotide |
|---|---|
| Distribution | Random across genome |
| Insertion rate | Higher than in P. falciparum |
| Confirmation rate | Majority confirmed by half-nested PCR |
Implementing this genetic approach requires specialized molecular tools and resources. Here are the key components researchers use to conduct piggyBac mutagenesis studies in malaria parasites:
| Research Tool | Function | Application in P. berghei Research |
|---|---|---|
| piggyBac donor plasmid | Contains transposon with drug resistance marker | Random integration into parasite genome |
| Helper plasmid | Provides transposase enzyme | Temporary expression of transposase for integration |
| Plasmodipur filters | Remove host white blood cells | Obtain pure parasite DNA for analysis |
| Illumina HiSeq system | High-throughput sequencing | Identify insertion sites across entire genome |
| BLAST and SOAP algorithms | Bioinformatics analysis | Map sequencing reads to reference genome |
Precise manipulation of parasite DNA
Specialized protocols for parasite culture
Advanced computational analysis
The implications of efficiently identifying piggyBac insertions extend far beyond basic science. By rapidly determining which genes have been disrupted in parasites with interesting phenotypes, researchers can:
By finding genes essential for parasite survival
By identifying genes crucial for mosquito stage development
By discovering genetic changes that confer resistance
By pinpointing genes encoding critical surface proteins
As research progresses, further improvements to the piggyBac system continue to emerge. Recent studies have demonstrated successful remobilization of piggyBac elements—moving insertions from one genomic location to another—in parasites that stably express the transposase 3 . This advancement means that with a single transfection, researchers can potentially generate multiple insertion events, bringing us closer to saturating mutagenesis where every non-essential gene in the parasite genome is disrupted.
While challenges remain—particularly in studying genes essential for blood-stage development, as disrupting these would kill the parasites before they could be studied—the combination of piggyBac mutagenesis and next-generation sequencing represents a powerful weapon in the scientific arsenal against malaria 4 .
"The availability of the technology for transposon-mediated random mutagenesis for P. berghei can be used to develop and apply large-scale forward genetic screens for analysing gene function" 3 .
This capability brings us closer to fully understanding the biology of this complex parasite and developing the novel interventions needed to finally control one of humanity's oldest diseases.