title: Recombination-mediated genetic engineering of Plasmodium berghei DNA.
abstract: DNA of Plasmodium berghei is difficult to manipulate in Escherichia coli by conventional restriction and ligation methods due to its high content of adenine and thymine (AT) nucleotides. This limits our ability to clone large genes and to generate complex vectors for modifying the parasite genome. We here describe a protocol for using lambda Red recombinase to modify inserts of a P. berghei genomic DNA library constructed in a linear, low-copy, phage-derived vector. The method uses primer extensions of 50 bp, which provide sufficient homology for an antibiotic resistance marker to recombine efficiently with a P. berghei genomic DNA insert in E. coli. In a subsequent in vitro Gateway reaction the bacterial marker is replaced with a cassette for selection in P. berghei. The insert is then released and used for transfection. The basic techniques we describe here can be adapted to generate highly efficient vectors for gene deletion, tagging, targeted mutagenesis, or genetic complementation with larger genomic regions.
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.
authors.forename: Claudia
authors.initials: C
authors.lastname: Pfander
authors.forename: Burcu
authors.initials: B
authors.lastname: Anar
authors.forename: Mathieu
authors.initials: M
authors.lastname: Brochet
authors.forename: Julian C
authors.initials: JC
authors.lastname: Rayner
authors.forename: Oliver
authors.initials: O
authors.lastname: Billker
mesh_terms: D003001:Cloning, Molecular; D016054:DNA, Protozoan; D018274:Electroporation / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D010957:Plasmids / Q000235:genetics; D010962:Plasmodium berghei / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D014169:Transformation, Bacterial
chemical_list: D016054:DNA, Protozoan