Plasmodium molecular tools

Plasmodium molecular tools are a set of methods for the genetic manipulation of Plasmodium genus of parasites. Plasmodium species have been difficult to scientifically study, partially due to the inability to use many standard biological techniques to genetically manipulate the organism. Recent research has sought to overcome these technical barriers in order to make the parasite more amenable to study. Below is a description of published methods of genetic control within the Plasmodium parasite.

Transformation

• Electroporation
• loaded RBCs

DNA level

Transcription regulation

• Tet-based transactivator system - ligand inducible control of gene transcription based upon the Tet system (P. falciparum)^[1]

Integration systems

• Rep20 mediated
• Bxb1 integrase - site-specific stable genetic integration into chromosomes mediated by mycobacteriophage Bxb1 integrase (P. falciparum)^[2]

Recombination systems

• Flp/FRT - induced site-specific recombination/mutagenesis using the yeast Flp/FRT system (P. berghei)^[3]

Transposon systems

• piggyBac - lepidopteran transposable element for random, efficient integration of DNA into genome (P. falciparum)^[4]

RNA level

• RNAi
• antisense
• self-cleaving ribozyme - A failed attempt to use an inducible self-cleaving ribozyme to control mRNA degradation of fused transcripts(P. falciparum)^[5]

Protein level

• FKBP destabilization domain - ligand (Shld1)-regulatable domain to promote degradation of fusion protein (P. falciparum)^[6]
• DHFR destabilization domain - ligand (Trimethoprim)-regulatable domain to promote degradation of fusion protein P. falciparum. By virtue of protein being tagged to a DHFR degradation domain from E. coli, as well as GFP and a HA-tag, protein levels can be regulated, cellular localization of the protein can be determined, and the protein can be purified from cultured parasites.^[7]

References

1. Meissner, M; Krejany, E; Gilson, PR; De Koning-Ward, TF; Soldati, D; Crabb, BS (2005). "Tetracycline analogue-regulated transgene expression in Plasmodium falciparum blood stages using Toxoplasma gondii transactivators". PNAS 102 (8): 2980–2985. doi:10.1073/pnas.0500112102. PMC 548799. PMID 15710888.
2. Nkrumah, LJ; Muhle, RA; Moura, PA; Ghosh, P; Hatfull, GF; Jacobs Jr, WR; Fidock, DA (2006). "Efficient site-specific integration in Plasmodium falciparum chromosomes mediated by mycobacteriophage Bxb1 integrase". Nature Methods 3 (8): 615–621. doi:10.1038/nmeth904. PMC 2943413. PMID 16862136.
3. Carvalho, TG; Thiberge, S; Sakamoto, H; Ménard, R (2004). "Conditional mutagenesis using site-specific recombination in Plasmodium berghei transactivators". PNAS 101 (41): 14931–14936. doi:10.1073/pnas.0404416101. PMC 522007. PMID 15465918.
4. Balu, B; Shoue, DA; Fraser Jr, MJ; Adams, JH (2005). "High-efficiency transformation of Plasmodium falciparum by the lepidopteran transposable element piggyBac". PNAS 102 (45): 16391–16396. doi:10.1073/pnas.0504679102. PMC 1275597. PMID 16260745.
5. Agop-Nersesian (2008). "Functional expression of ribozymes in Apicomplexa: Towards exogenous control of gene expression by inducible RNA-cleavage". International Journal for Parasitology. doi:10.1016/j.ijpara.2007.10.015.
6. Armstrong, CM; Goldberg, DE (2007). "An FKBP destabilization domain modulates protein levels in Plasmodium falciparum". Nature Methods 4 (12): 1007–1009. doi:10.1038/nmeth1132. PMID 17994030.
7. Muralidharan, V; Goldberg, DE (2011). "Asparagine repeat function in a Plasmodium falciparum protein assessed via a regulatable fluorescent affinity tag". PNAS 108 (11): 4411–4416. doi:10.1073/pnas.1018449108. PMC 3060247. PMID 21368162.