原创力文档removal of heterologous sequences from plasmodium falciparum mutants using flpe-recombinase除使用flpe-recombinase恶性疟原虫的不同的序列突变体.pdfVIP
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removal of heterologous sequences from plasmodium falciparum mutants using flpe-recombinase除使用flpe-recombinase恶性疟原虫的不同的序列突变体
Removal of Heterologous Sequences from Plasmodium
falciparum Mutants Using FLPe-Recombinase
1 1 2 1 2
Ben C. L. van Schaijk *, Martijn W. Vos , Chris J. Janse , Robert W. Sauerwein , Shahid M. Khan *
1 Department of Medical Microbiology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands, 2 Leiden Malaria Research Group, Department of
Parasitology, Center of Infectious Diseases, Leiden University Medical Centre, Leiden, The Netherlands
Abstract
Genetically-modified mutants are now indispensable Plasmodium gene-function reagents, which are also being pursued as
genetically attenuated parasite vaccines. Currently, the generation of transgenic malaria-parasites requires the use of drug-
resistance markers. Here we present the development of an FRT/FLP-recombinase system that enables the generation of
transgenic parasites free of resistance genes. We demonstrate in the human malaria parasite, P. falciparum, the complete
and efficient removal of the introduced resistance gene. We targeted two neighbouring genes, p52 and p36, using a
construct that has a selectable marker cassette flanked by FRT-sequences. This permitted the subsequent removal of the
selectable marker cassette by transient transfection of a plasmid that expressed a 37uC thermostable and enhanced FLP-
recombinase. This method of removing heterologous DNA sequences from the genome opens up new possibilities in
Plasmodium research to sequentially target multiple genes and for using genetically-modified parasites as live, attenuated
malaria vaccines.
Citation: van Schaijk BCL, Vos MW, Janse CJ, Sauerwein RW, Khan SM (2010) Removal of Heterologous Sequences from Plasmodium falciparum Mutants Using
FLPe-R
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