Pfander Claudia, Anar Burcu, Brochet Mathieu, Rayner Julian C, Billker Oliver
Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.
Methods Mol Biol. 2013;923:127-38. doi: 10.1007/978-1-62703-026-7_8.
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.
由于伯氏疟原虫的DNA中腺嘌呤和胸腺嘧啶（AT）核苷酸含量很高，采用传统的限制性内切酶和连接方法很难在大肠杆菌中对其进行操作。这限制了我们克隆大基因以及构建用于修饰寄生虫基因组的复杂载体的能力。我们在此描述了一种使用λ Red重组酶来修饰在一种线性、低拷贝、噬菌体衍生载体中构建的伯氏疟原虫基因组DNA文库插入片段的方案。该方法使用50 bp的引物延伸，这为抗生素抗性标记与大肠杆菌中的伯氏疟原虫基因组DNA插入片段高效重组提供了足够的同源性。在随后的体外Gateway反应中，细菌标记被用于在伯氏疟原虫中进行选择的盒式结构所取代。然后释放插入片段并用于转染。我们在此描述的基本技术可加以调整，以生成用于基因缺失、标记、靶向诱变或与更大基因组区域进行基因互补的高效载体。
