Development and application of a positive-negative selectable marker system for use in reverse genetics in Plasmodium.
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.
Department of Parasitology, Centre of Infectious Diseases, Leiden University Medical Centre, The Netherlands.
Joanna A M
JA
Braks
Blandine
B
Franke-Fayard
Hans
H
Kroeze
Chris J
CJ
Janse
Andrew P
AP
Waters
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000378:metabolism; D043525:Cytosine Deaminase / Q000235:genetics; D005437:Flucytosine / Q000494:pharmacology; D050939:Gene Fusion; D018390:Gene Targeting / Q000379:methods*; D005819:Genetic Markers; D005822:Genetic Vectors; D051379:Mice; D009154:Mutation; D010430:Pentosyltransferases / Q000235:genetics; D010641:Phenotype; D010962:Plasmodium berghei / Q000187:drug effects / Q000235:genetics*; D015800:Protozoan Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis; D011995:Recombination, Genetic; D013937:Thymidine Kinase / Q000235:genetics; D014170:Transformation, Genetic
D005819:Genetic Markers; C422062:P48 protein, Plasmodium berghei; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D005437:Flucytosine; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase; D013937:Thymidine Kinase; D043525:Cytosine Deaminase