Towards systematic identification of Plasmodium essential genes by transposon shuttle mutagenesis.
After the deciphering of the genome sequences of several Plasmodium species, efforts must turn to elucidating gene function and identifying essential gene products. However, random approaches are lacking and gene targeting is inefficient in Plasmodium. Here, we established shuttle transposon mutagenesis in Plasmodium berghei. We constructed a mini-Tn5 derivative that can transpose into parasite genes cloned in Escherichia coli, providing an efficient means of generating knockout fragments. A 10(4)-fold increase in frequencies of double-crossover homologous recombination in the parasite using a new electroporation technology permits to reproducibly generate pools of distinct mutants after transfection with mini-Tn5-interrupted sequences. The procedure opens the way to the systematic identification of essential genes in Plasmodium.
Sakamoto
Hiroshi
H
Unité de Biologie et Génétique du Paludisme, Institut Pasteur, 28 Rue du Dr Roux, 75724 Paris cedex 15, France.
Thiberge
Sabine
S
Akerman
Susan
S
Janse
Chris J
CJ
Carvalho
Teresa Gil
TG
Ménard
Robert
R
D000818:Animals; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D016054:DNA, Protozoan / Q000235:genetics; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics; D017125:Genes, Protozoan; D016254:Mutagenesis, Insertional / Q000379:methods*; D010962:Plasmodium berghei / Q000235:genetics*; D011995:Recombination, Genetic; D014162:Transfection
D004251:DNA Transposable Elements; D016054:DNA, Protozoan