Development and application of a positive-negative selectable marker system for use in reverse genetics in Plasmodium
Open Access
- 6 March 2006
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 34 (5) , e39
- https://doi.org/10.1093/nar/gnj033
Abstract
A limitation of transfection of malaria parasites is the availability of only a low number of positive selectable markers for selection of transformed mutants. This is exacerbated for the rodent parasite Plasmodium berghei as selection of mutants is performed in vivo in laboratory rodents. We here report the development and application of a negative selection system based upon transgenic expression of a bifunctional protein (yFCU) combining yeast cytosine deaminase and uridyl phosphoribosyl transferase (UPRT) activity in P.berghei followed by in vivo selection with the prodrug 5-fluorocytosine (5-FC). The combination of yfcu and a positive selectable marker was used to first achieve positive selection of mutant parasites with a disrupted gene in a conventional manner. Thereafter through negative selection using 5-FC, mutants were selected where the disrupted gene had been restored to its original configuration as a result of the excision of the selectable markers from the genome through homologous recombination. This procedure was carried out for a Plasmodium gene (p48/45) encoding a protein involved in fertilization, the function of which had been previously implied through gene disruption alone. Such reversible recombination can therefore be employed for both the rapid analysis of the phenotype by targeted disruption of a gene and further associate phenotype and function by genotype restoration through the use of a single plasmid and a single positive selectable marker. Furthermore the negative selection system may also be adapted to facilitate other procedures such as 'Hit and Run' and 'vector recycling' which in principle will allow unlimited manipulation of a single parasite clone. This is the first demonstration of the general use of yFCU in combination with a positive selectable marker in reverse genetics approaches and it should be possible to adapt its use to many other biological systems.Keywords
This publication has 33 references indexed in Scilit:
- Development of a recombinant Leishmania major strain sensitive to ganciclovir and 5-fluorocytosine for use as a live vaccine challenge in clinical trialsVaccine, 2005
- A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycleMolecular and Biochemical Parasitology, 2004
- Calcium and a Calcium-Dependent Protein Kinase Regulate Gamete Formation and Mosquito Transmission in a Malaria ParasiteCell, 2004
- Negative selection of Plasmodiumfalciparum reveals targeted gene deletion by double crossover recombinationInternational Journal for Parasitology, 2002
- Puromycin-N-acetyltransferase as a selectable marker for use in Plasmodium falciparumMolecular and Biochemical Parasitology, 2001
- Functional Equivalence of Structurally Distinct Ribosomes in the Malaria Parasite, Plasmodium bergheiPublished by Elsevier ,2001
- A Central Role for P48/45 in Malaria Parasite Male Gamete FertilityCell, 2001
- The selectable marker human dihydrofolate reductase enables sequential genetic manipulation of the Plasmodium berghei genomeMolecular and Biochemical Parasitology, 2000
- Enzyme/Prodrug Therapy for Head and Neck Cancer Using a Catalytically Superior Cytosine DeaminaseHuman Gene Therapy, 1999
- Comparison of in vivo and in vitro antimalarial activity of artemisinin, dihydroartemisinin and sodium artesunate in the Plasmodium berghei-rodent modelInternational Journal for Parasitology, 1994