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Nucleic Acids Research 2006 34(5):e39; doi:10.1093/nar/gnj033
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Published online 14 March 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
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Development and application of a positive–negative selectable marker system for use in reverse genetics in Plasmodium

Joanna A. M. Braks, Blandine Franke-Fayard, Hans Kroeze, Chris J. Janse and Andrew P. Waters*

Department of Parasitology, Centre of Infectious Diseases, Leiden University Medical Centre (LUMC) The Netherlands

*To whom correspondence should be addressed. Tel: +31 71 5265069; Fax: +31 71 5266907; Email: waters{at}lumc.nl

Received December 6, 2005. Revised February 4, 2006. Accepted February 4, 2006.

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.


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[Abstract] [Full Text] [PDF]


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